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, TiO₂ 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 Mn₂O₃ NPs 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, CeO₂ and TiO₂ 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.

Appendix: Equations

To normalize the absorbance values using the normalization factors:

$$y = A_{520} \;{\text{of}}\;{\text{reduced}}\;{\text{reference}} - A_{520} \; {\text{of}}\;{\text{sample}}$$

To take into account NP interference:

$$A = A_{\text{sample}} - A_{\text{NPs}}$$

Calculation of the oxidized/reduced cytochrome c value k.

$$k = \frac{{A_{{520{\text{nm}}}} + A_{{549{\text{nm}}}} - A_{{532{\text{nm }} }} }}{{A_{{520{\text{nm}}}} }}$$

(1)

Calculation of the percentage of reduced cytochrome c in solution.

$$\delta = \frac{{k_{\text{sample}} - k_{{{\text{oxidized}}\;{\text{reference}}}} }}{{k_{{{\text{reduced}}\;{\text{reference}}}} - k_{{{\text{oxidized}}\;{\text{reference}}}} }} \times 100$$

(2)

Calculation of percent of reduced cytochrome c in comparison with the reduced cytochrome c control.

$${\text{Reduced}}\;{\text{cyt}}\;c \,\left( {\% \;{\text{of}}\;{\text{control}}} \right) = \frac{{\delta_{\text{sample}} \left( t \right)}}{{\delta_{{{\text{reduced}}\;{\text{cyt}}\;c\;{\text{control}}}} \left( t \right)}} \times 100$$

(3)

reduced reference = fully reduced cytochrome c at t ₀, oxidized reference = fully oxidized cytochrome c at t ₀, reduced control = fully reduced cytochrome c at t.