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

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.

High-Dose Exposure to Polymer-Coated Iron Oxide Nanoparticles Elicits Autophagy-Dependent Ferroptosis in Susceptible Cancer Cells

“…The mixture was then purified by washing with a phosphate-buffered solution and concentrated (Pellicon XL, 500 kDa, Merck, Darmstadt, Germany) to a final volume of 150 mL [0.3% (w/w) in Fe 3 O 4 ]; the mixture was then filtered through a polyethersulfone membrane (0.22 µm, Millipore Sterivex™). The TEM and DLS analyses of Fe 3 O 4 -PEG-PLGA nanoparticles were reported previously by us [18].…”

“…THP-1 cells (differentiated into macrophage-like cells) and HL-60 cells (differentiated into neutrophil-like cells) were thus included. The nanoparticles were characterized with respect to the physicochemical properties in a previous study [18]. The primary particle size (by TEM) was 12 ± 4 nm.…”

Nanomaterial genotoxicity evaluation using the high-throughput p53-binding protein 1 (53BP1) assay

“…Although flow cytometry is not as high throughput and not as classically used as spectrofluorometry, it may make possible the detection of low-grade elevation of ROS levels that could explain the genotoxic damage caused by these TiO2 NPs. In addition, a recent study published by the BIORIMA consortium and performed with the same TiO2 NPs (NM101), using the U2OS-nuclear factor (erythroid-derived 2)-like 2 (NRF2) reporter cell line, did not show any activation of the NRF2 pathway [121], which is usually activated when cells undergo significant oxidative stress. Therefore, TiO2 NMs might generate some reactive species that are not reactive towards the DHR123 probe, or at levels that are too low to be detected by this assay, and might not generate sufficient oxidative stress to trigger the NRF2 pathway but might travel to the cell nucleus and damage DNA.…”

3D model of the bronchial epithelial barrier to study repeated exposure to xenobiotics: Application to silver nanoparticles

“…A lower induction was observed after the second exposure to AgNO 3 in contrary to AgNPs, which suggests that AgNP dissolution is involved in the delayed metal stress response observed in epithelial cells following multiple exposure. We have shown previously by SAXS analysis that AgNP dissolution in MEM is very limited after incubation for 18 days at 37°C (Martin et al 2022). Therefore, it is likely that AgNP dissolution occurs after endocytosis in the phagolysosomes observed by TEM, where dissolution would be favoured by low pH.…”

“…Two populations were identi ed with average hydrodynamic diameters of 4.5 ± 0.8 and 14.1 ± 0.5 nm respectively, in agreement with JRC results. The stability of AgNPs in Eagle's Minimum Essential Medium (MEM, Gibco, Life technologies, Courtaboeuf, France) supplemented with 4% fetal bovine serum (FBS, Sigma-Aldrich, Saint-Quentin, France) was investigated in a previous study by Small X-Ray Angle Scattering (SAXS) to determine if dissolution occurs during the experiment time (Martin et al 2022). An average diameter of 15.4 ± 0.2 nm was measured by SAXS and no dissolution was observed after incubation in at 37°C for 18 days.…”