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, MnO, Cu, Ag, BaSO, CeO, TiO and 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 MnO NPs induced a strong oxidation of cytochrome c and a dose-dependent cytotoxicity, pro-inflammatory response and antioxidant enzyme expression. BaSO, which presented no intrinsic oxidative potential, had no cellular effects. Nevertheless, CeO and TiO NPs 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.

Surface reactivity measurements as required for grouping and read-across: An advanced FRAS protocol

“…Based on earlier report of both FRAS assay [7] and cytochrome c assay [10] and band gap analysis [11], Mn2O3 is recurring as the most reactive, yet insoluble material. Hence, we chose to establish Mn2O3 as positive control.…”

“…Hence, we chose to establish Mn2O3 as positive control. We use the same batch as in [10], sourced from Skyspring Nanomaterials, with primary particle diameters ranging from 20 to 100 nm (TEM) and a specific surface area of 19.9 m²/g (BET). [10] As expected, Mn2O3 induces maximum antioxidant damage already at low dose ( Figure 4, red line), and levels off into a plateau at higher doses.…”

“…We use the same batch as in [10], sourced from Skyspring Nanomaterials, with primary particle diameters ranging from 20 to 100 nm (TEM) and a specific surface area of 19.9 m²/g (BET). [10] As expected, Mn2O3 induces maximum antioxidant damage already at low dose ( Figure 4, red line), and levels off into a plateau at higher doses. The maximum sBOD of 1610 ± 4 nmol TEU/ m² (equivalent to mBOD of 32.0 ± 0.09 nmol TEU/ mg) is reached at a surface dose of 244.5 m²/L (corresponding to mass concentration of 4.9 g/L), generating a BOD of 156,680 ± 435 mM TEU.…”

“…Both also agree on excellent reproducibility of Mn2O3 reactivity (<1% standard deviation in our triplicates). Mn2O3 is thus chosen as our positive control, as established also for the cytochrome-c [10] and bandgap assays. [11] 3.2.1.…”

“…It has frequently been identified in in vitro and in vivo studies [2,3] and different assays have been developed for this purpose. [4][5][6][7][8][9][10][11] Previously, we have investigated a representative panel of 32 ENMs including functionalized amorphous silica (6 nanoforms), zirconium dioxide (4), silver (4), titanium dioxide (3), zinc oxide (2), multiwalled carbon nanotubes (3), barium sulfate and AlOOH. Surface reactivities of all were studied in a cell-free system by electron spin resonance (ESR) spectroscopy.…”