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

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.

Cytotoxic Effects Caused by Functionalized Carbon Nanotube in Murine Macrophages.

“…The results showed a significant increase in nitric oxide (NO) and ROS production in the two periods tested, but the ROS production in 24 h was even higher when compared to the results in 48 hours of exposure. The ROS quantification data corroborate the findings by Sabido et al, who tested CNTs in RAW murine macrophages and observed an increase in ROS production initially and subsequently decrease in the production of this compound after exposure for longer periods of CNTs, characterizing oxidative stress as a rapid and transitory process [27]. Oxidative stress has the ability to trigger and maintain inflammation mechanisms, generating possible toxic effects on cells, as well as overproduction of ROS can act directly on biological structures causing damage to cellular functions [27][28][29].…”

Octacalcium Phosphate for Bone Tissue Engineering: Synthesis, Modification, and In Vitro Biocompatibility Assessment

“…To evaluate the production of reactive oxygen species (ROS) in cells, after 96 h of co-incubation with OCP and OCP-Sr, the cells were washed three times with PBS solution, then detached from the surface of the culture plastic using 0.05% trypsin-EDTA solution and stained with 20 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), (Sigma-Aldrich, St. Louis, MO, USA) for 15 min in a CO 2 incubator [ 50 ]. As a control, cells were incubated with 1 mM hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) for 20 min.…”

Carbon nanotube nanocomposite scaffolds: advances in fabrication and applications for tissue regeneration and cancer therapy

“…It seems that increase in reactive oxygen species (ROS) is the primary pathway through which CNT accumulation induces oxidative stress, which in turn leads to damage in genetic material and alterations in the cell cycle ( Cheng et al, 2011 ; Azad et al, 2013 ). Specifically, SWCNT leads to hydroxyl radical production, activating activator protein-1 (AP-1) pathways, mitogen activated protein kinase (MAPK) pathways, and nuclear factor-kB (NF-kB) pathways of inflammatory response ( Sabido et al, 2020 ). This effect is reduced for MWCNTs, as the greater delocalization area increases MWCNT’s scavenging capacity.…”