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Quantitative Flow Cytometric Evaluation of Oxidative Stress and Mitochondrial Impairment in RAW 264.7 Macrophages after Exposure to Pristine, Acid Functionalized, or Annealed Carbon Nanotubes

1
Inserm U1059 SAINBIOSE, équipe DVH/PIB, Université Jean Monnet, Faculté de Médecine, F-42270 Saint-Etienne, France
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Université Lyon, F-42270 Saint-Etienne, France
3
Centre Commun de Cytométrie en Flux, F-42270 Saint-Etienne, France
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Ecole Nationale Supérieure des Mines, SPIN, CNRS: UMR 5307, LGF, F-42023 Saint-Etienne, France
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Mines Saint-Etienne, Univ Lyon, Univ Jean Monnet, INSERM, U1059 Sainbiose, Centre CIS, F-42023 Saint-Etienne, France
6
Dipartimento di Chimica and ‘G. Scansetti’ Interdepartmental Center for Studies on Asbestos and other Toxic Particulates, Università di Torino, 10125, Torino, Italy
*
Authors to whom correspondence should be addressed.
Nanomaterials 2020, 10(2), 319; https://doi.org/10.3390/nano10020319
Received: 28 December 2019 / Revised: 4 February 2020 / Accepted: 7 February 2020 / Published: 13 February 2020
Conventional nanotoxicological assays are subjected to various interferences with nanoparticles and especially carbon nanotubes. A multiparametric flow cytometry (FCM) methodology was developed here as an alternative to quantify oxidative stress, mitochondrial impairment, and later cytotoxic and genotoxic events. The experiments were conducted on RAW264.7 macrophages, exposed for 90 min or 24 h-exposure with three types of multiwalled carbon nanotubes (MWCNTs): pristine (Nanocyl™ CNT), acid functionalized (CNTf), or annealed treatment (CNTa). An original combination of reactive oxygen species (ROS) probes allowed the simultaneous quantifications of broad-spectrum ROS, superoxide anion (O2•−), and hydroxyl radical (•OH). All MWCNTs types induced a slight increase of broad ROS levels regardless of earlier antioxidant catalase activity. CNTf strongly stimulated the O2•− production. The •OH production was downregulated for all MWCNTs due to their scavenging capacity. The latter was quantified in a cell-free system by electron paramagnetic resonance spectroscopy (EPR). Further FCM-based assessment revealed early biological damages with a mitochondrial membrane potential collapse, followed by late cytotoxicity with chromatin decondensation. The combined evaluation by FCM analysis and cell-free techniques led to a better understanding of the impacts of MWCNTs surface treatments on the oxidative stress and related biological response. View Full-Text
Keywords: carbon nanotubes; acid functionalization; annealing treatment; oxidative stress; mitochondrial membrane potential collapse; cytotoxicity; chromatin decondensation, scavenging capacity; flow cytometry and spin trapping in-cell free system carbon nanotubes; acid functionalization; annealing treatment; oxidative stress; mitochondrial membrane potential collapse; cytotoxicity; chromatin decondensation, scavenging capacity; flow cytometry and spin trapping in-cell free system
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MDPI and ACS Style

Sabido, O.; Figarol, A.; Klein, J.-P.; Bin, V.; Forest, V.; Pourchez, J.; Fubini, B.; Cottier, M.; Tomatis, M.; Boudard, D. Quantitative Flow Cytometric Evaluation of Oxidative Stress and Mitochondrial Impairment in RAW 264.7 Macrophages after Exposure to Pristine, Acid Functionalized, or Annealed Carbon Nanotubes. Nanomaterials 2020, 10, 319.

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