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.

Odile Sabido,Valérie Bin,Jérémie Pourchez,Bice Fubini,Jean-Philippe Klein,Maura Tomatis,Agathe Figarol,Valérie Forest,Michèle Cottier,Delphine Boudard

doi:10.3390/nano10020319

Abstract

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.

Highlights

The use of nanomaterials in many industrial fields has tremendously raised over the last two decades; nanotechnologies represent a massive worldwide investment [1,2]
We developed here a quantitative multiparametric flow cytometry (FCM) approach to investigate early alterations such as oxidative stress generation and mitochondrial potential alteration, and late events such as cytotoxicity and genotoxicity
For the DCFH-DA quantification after 90 min, the levels of intracellular reactive oxygen species (ROS) in the cells exposed to a 120 μg·mL−1 dose increased by twice with respect to the negative control, without distinction of Multiwalled carbon nanotubes (MWCNTs) types

Summary

Introduction

The use of nanomaterials in many industrial fields has tremendously raised over the last two decades; nanotechnologies represent a massive worldwide investment [1,2]. Carbon nanotubes present unique properties in terms of strength, electrical conductivity, or heat conduction. They are suitable for applications in high-performance materials and recently considered for use in biomedical research [3,4,5,6,7,8]. There is growing evidence concerning the induction by MWCNTs of three main toxicological mechanisms: oxidative stress, inflammation, and cytotoxicity/genotoxicity [5,12,21,23,24,25,26]. Oxidative stress triggers and maintains inflammation, and it eventually induces cytotoxic and genotoxic effects [6,12,25,28,29]. The overproduction of ROS can effectively affect biological structures and lead to cell function damages [6,16,30]

Methods

Results

Discussion

Conclusion