title: Assessment of the oxidative potential of nanoparticles by the cytochrome c assay: assay improvement and development of a high-throughput method to predict the toxicity of nanoparticles.
abstract: Oxidative stress has increasingly been demonstrated as playing a key role in the biological response induced by nanoparticles (NPs). The acellular cytochrome c oxidation assay has been proposed to determine the intrinsic oxidant-generating capacity of NPs. Yet, there is a need to improve this method to allow a rapid screening to classify NPs in terms of toxicity. We adapted the cytochrome c assay to take into account NP interference, to improve its sensitivity and to develop a high-throughput method. The intrinsic oxidative ability of a panel of NPs (carbon black, Mn2O3, Cu, Ag, BaSO4, CeO2, TiO2and ZnO) was measured with this enhanced test and compared to other acellular redox assays. To assess whether their oxidative potential correlates with cellular responses, we studied the effect of insoluble NPs on the human bronchial epithelial cell line NCI-H292 by measuring the cytotoxicity (WST-1 assay), pro-inflammatory response (IL-8 cytokine production and expression) and antioxidant defense induction (SOD2 and HO-1 expression). The adapted cytochrome c assay had a greatly increased sensitivity allowing the ranking of NPs in terms of their oxidative potential by using the developed high-throughput technique. Besides, a high oxidative potential revealed to be predictive for toxic effects as Mn2O3NPs induced a strong oxidation of cytochrome c and a dose-dependent cytotoxicity, pro-inflammatory response and antioxidant enzyme expression. BaSO4, which presented no intrinsic oxidative potential, had no cellular effects. Nevertheless, CeO2and TiO2NPs demonstrated no acellular oxidant-generating capacity but induced moderate cellular responses. In conclusion, the novel cytochrome c oxidation assay could be used for high-throughput screening of the intrinsic oxidative potential of NPs. However, acellular redox assays may not be sufficient to discriminate among low-toxicity NPs, and additional tests are thus needed.
authors.affiliation: Unit of Functional and Adaptive Biology (BFA), Lab of Molecular and Cellular Responses to Xenobiotics, UMR CNRS 8251, Univ-Paris-Diderot (Sorbonne Paris Cité), 5 rue Thomas Mann, 75205, Paris Cedex 13, France.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Delaval
authors.affiliation: Material Physics GMC/R, BASF SE, 67056, Ludwigshafen am Rhein, Germany.
authors.forename: Wendel
authors.initials: W
authors.lastname: Wohlleben
authors.affiliation: Experimental Toxicology and Ecology Department, BASF SE, 67056, Ludwigshafen am Rhein, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Landsiedel
authors.affiliation: Unit of Functional and Adaptive Biology (BFA), Lab of Molecular and Cellular Responses to Xenobiotics, UMR CNRS 8251, Univ-Paris-Diderot (Sorbonne Paris Cité), 5 rue Thomas Mann, 75205, Paris Cedex 13, France.
authors.forename: Armelle
authors.initials: A
authors.lastname: Baeza-Squiban
authors.affiliation: Unit of Functional and Adaptive Biology (BFA), Lab of Molecular and Cellular Responses to Xenobiotics, UMR CNRS 8251, Univ-Paris-Diderot (Sorbonne Paris Cité), 5 rue Thomas Mann, 75205, Paris Cedex 13, France. boland@univ-paris-diderot.fr.
authors.forename: Sonja
authors.initials: S
authors.lastname: Boland
mesh_terms: D000818:Animals; D001980:Bronchi / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D055598:Chemical Phenomena; D045304:Cytochromes c / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006736:Horses; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity*; D016877:Oxidants / Q000737:chemistry / Q000633:toxicity*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D010316:Particle Size; D017382:Reactive Oxygen Species / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D020545:Respiratory Mucosa / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D013499:Surface Properties; D018675:Toxicity Tests
chemical_list: D007202:Indicators and Reagents; D016877:Oxidants; D017382:Reactive Oxygen Species; D045304:Cytochromes c
keywords: Barium sulfate; Carbon black; Cell culture; Cerium; Copper; In vitro; Lung; Manganese; Nanomaterial; Nanotoxicity; Nanotoxicology; Oxide; Pulmonary; Reactive oxygen species; Silver; Titanium; Zinc