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

Martin, Sebastin; de Haan, Laura; Miro Estruch, Ignacio; Eder, Kai Moritz; Marzi, Anne; Schnekenburger, J�rgen; Blosi, Magda; Costa, Anna; Antonello, Giulia; Bergamaschi, Enrico; Riganti, Chiara; Beal, David; Carri�re, Marie; Tach�, Olivier; Hutchison, Gary; Malone, Eva; Young, Lesley; Campagnolo, Luisa; La Civita, Fabio; Pietroiusti, Antonio; Devineau, St�phanie; Baeza, Armelle; Boland, Sonja; Zong, Cai; Ichihara, Gaku; Fadeel, Bengt; Bouwmeester, Hans

Authors

Sebastin Martin

Laura de Haan

Ignacio Miro Estruch

Kai Moritz Eder

Anne Marzi

J�rgen Schnekenburger

Magda Blosi

Anna Costa

Giulia Antonello

Enrico Bergamaschi

Chiara Riganti

David Beal

Marie Carri�re

Olivier Tach�

Luisa Campagnolo

Fabio La Civita

Antonio Pietroiusti

St�phanie Devineau

Armelle Baeza

Sonja Boland

Cai Zong

Gaku Ichihara

Bengt Fadeel

Hans Bouwmeester



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.

Citation

Martin, S., de Haan, L., Miro Estruch, I., Eder, K. M., Marzi, A., Schnekenburger, J., …Bouwmeester, H. (2022). Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials. Frontiers in Toxicology, 4, Article 974429. https://doi.org/10.3389/ftox.2022.974429

Journal Article Type Article
Acceptance Date Aug 9, 2022
Online Publication Date Sep 5, 2022
Publication Date 2022
Deposit Date Oct 6, 2022
Publicly Available Date Oct 6, 2022
Journal Frontiers in Toxicology
Publisher Frontiers Media
Peer Reviewed Peer Reviewed
Volume 4
Article Number 974429
DOI https://doi.org/10.3389/ftox.2022.974429
Keywords Nrf2, nanomaterial, interlaboratory validation, oxidative stress, nanotoxicology
Public URL http://researchrepository.napier.ac.uk/Output/2926540

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Publisher Licence URL
http://creativecommons.org/licenses/by/4.0/

Copyright Statement
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.




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