Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials

Sebastin Martin (1) , Laura de Haan (2) , Ignacio Miro Estruch (2) , Kai Moritz Eder (3) , Anne Marzi (3) , Jürgen Schnekenburger (3) , Magda Blosi (4) , Anna Costa (4) , Giulia Antonello (5) , Enrico Bergamaschi (5) , Chiara Riganti (5) , David Beal (6) , Marie Carrière (6, 7) , Olivier Taché (8) , Gary Hutchison (9) , Eva Malone (9) , Lesley Young (9) , Luisa Campagnolo (10) , Fabio La Civita (10) , Antonio Pietroiusti (10) , Stéphanie Devineau (10) , Armelle Baeza (10) , Sonja Boland (10) , Cai Zong (11) , Gaku Ichihara (11) , Bengt Fadeel (12) , Hans Bouwmeester (2)

1 Karolinska Institute
2 WUR - Wageningen University and Research [Wageningen]
3 WWU - Westfälische Wilhelms-Universität Münster = University of Münster
4 CNR | ISTEC - CNR Istituto di Scienza e Tecnologia dei Materiali Ceramici [Faenza]
5 UNITO - Università degli studi di Torino = University of Turin
6 SYMMES - SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé
7 CIBEST - Chimie Interface Biologie pour l’Environnement, la Santé et la Toxicologie
8 LIONS - Laboratoire Interdisciplinaire sur l'Organisation Nanométrique et Supramoléculaire
9 Edinburgh Napier University
10 BFA (UMR_8251 / U1133) - Unité de Biologie Fonctionnelle et Adaptative
11 Tokyo University of Science [Tokyo]
12 Karolinska Institutet [Stockholm]

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 Fe 3 O 4 -PEG-PLGA nanomaterials while Ag and TiO 2 “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. Fe 3 O 4 -PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO 2 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.

Mots clés: Nrf2; nanomaterial; interlaboratory validation; oxidative stress; nanotoxicology

Domaines: Sciences du Vivant [q-bio]; Matériaux

Sebastin Martin, Laura de Haan, Ignacio Miro Estruch, Kai Moritz Eder, Anne Marzi, et al.. Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials. Frontiers in Toxicology, 2022, 4, ⟨10.3389/ftox.2022.974429⟩. ⟨cea-03789240⟩