Figure S1
A.
0.4
B.
Cyt c + Mn 2O3 NPs 100µg/ml
Cyt c + Mn 2O3 NPs 50µg/ml
Cyt c + Mn 2O3 NPs 25µg/ml
Mn2O3 NPs 100µg/ml
Cyt c + CeO 2 NPs 100µg/ml
CeO2 NPs 100µg/ml
Mn2O3 NPs 50µg/ml
Cyt c + CeO 2 NPs 50µg/ml
CeO2 NPs 50µg/ml
Mn2O3 NPs 25µg/ml
Cyt c + CeO 2 NPs 25µg/ml
CeO2 NPs 25µg/ml
0.8
0.6
Absorbance
Absorbance
0.3
0.2
0.1
0.0
450
0.4
0.2
500
550
600
0.0
450
650
500
Wavelength (nm)
C.
1.5
550
600
650
Wavelength (nm)
D.
Cyt c + TiO 2 NPs 100µg/ml
TiO2 NPs 100µg/ml
Cyt c + BaSO 4 NPs 100µg/ml
BaSO4 NPs 100µg/ml
Cyt c + TiO 2 NPs 50µg/ml
TiO2 NPs 50µg/ml
Cyt c + BaSO 4 NPs 50µg/ml
BaSO4 NPs 50µg/ml
Cyt c + TiO 2 NPs 25µg/ml
TiO2 NPs 25µg/ml
Cyt c + BaSO 4 NPs 25µg/ml
BaSO4 NPs 25µg/ml
0.4
1.0
Absorbance
Absorbance
0.3
0.5
0.2
0.1
0.0
450
500
550
600
0.0
450
650
500
Wavelength (nm)
E.
Absorbance
0.20
Cu NPs 100µg/ml
Cyt c + Cu NPs 50µg/ml
Cu NPs 50µg/ml
Cyt c + Cu NPs 25µg/ml
Cu NPs 25µg/ml
0.25
0.20
0.15
0.10
0.05
0.00
450
ZnO NPs 100µg/ml
Cyt c + ZnO NPs 50µg/ml
ZnO NPs 50µg/ml
Cyt c + ZnO NPs 25µg/ml
ZnO NPs 25µg/ml
0.15
0.10
0.05
500
550
600
0.00
450
650
500
Cyt c + CB NPs 25µg/ml
0.25
CB NPs 100µg/ml
CB NPs 50µg/ml
CB NPs 25µg/ml
0.20
Absorbance
Absorbance
600
650
H.
Cyt c + CB NPs 100µg/ml
Cyt c + CB NPs 50µg/ml
0.15
0.10
0.05
0.00
450
550
Wavelength (nm)
G.
0.20
650
Cyt c + ZnO NPs 100µg/ml
Wavelength (nm)
0.25
600
F.
Cyt c + Cu NPs 100µg/ml
Absorbance
0.25
550
Wavelength (nm)
Cyt c + Ag NPs 100µg/ml
Ag NPs 100µg/ml
Cyt c + Ag NPs 50µg/ml
Ag NPs 50µg/ml
Cyt c + Ag NPs 25µg/ml
Ag NPs 25µg/ml
0.15
0.10
0.05
500
550
Wavelength (nm)
600
650
0.00
450
500
550
Wavelength (nm)
600
650
Figure S1. Absorption spectrum of NPs incubated with or without 30 µM of reduced horse-heart cytochrome c. 25, 50
and 100 µg/ml of Mn2O3 NPs (A.), CeO2 NPs (B.), TiO2 NPs (C.), BaSO4 NPs (D.), Cu NPs (E.), ZnO NPs (F.), CB NPs (G.)
and Ag NPs (H.) were incubated with 30 µM reduced cytochrome c in 0.01 M of potassium phosphate buffer at pH 7.4.
Absorption spectra within the q-band (450 nm - 650 nm) was directly determined by absorbance measurement.
Figure S2
1.5
Cyt c w/o centrigugation
Mn2O3 NPs w/o
CeO2 NPs w/o
TiO 2 NPs w/o
BaSO 4 NPs w/o
Cyt c w centrifugation
Mn2O3 NPs w
CeO2 NPs w
TiO 2 NPs w
BaSO 4 NPs w
Absorbance
1.0
0.5
0.0
450
500
550
600
650
Wavelength (nm)
Figure S2. Dealing with interference of NPs with the cytochrome c oxidation measurement by centrifugation. 20 µM of
cytochrome c were incubated with or without 100 µg/ml of NPs. Samples were centrifuged 5 minutes at 3000g. Absorption
spectra of cyt c with (dotted lines) or without (full lines) NPs measured before (w/o, dark lines) and after (w, grey lines)
centrifugation.
DTT depletion assay
The progressive oxidation of the dithiothreitol (DTT) by NPs was determined according to Cho and al.
(Cho et al. 2005) by adding DTNB (5,5 '-dithiobis-dithiobis (2-nitrobenzoic acid)). Briefly, oxygen was removed
from the solutions with a vacuum pump during 3 hours minimum. To establish a DTT (Sigma-Aldrich) standard
curve, 0 to 250 µM DTT solutions were prepared in HEPES buffer 15 mM [4-(2-hydroxyethyl)-1piperazineethanesulfonic acid], Sigma-Aldrich) pH 7.4 and deposited in a 96-well plate. Particle suspensions
were diluted in 15 mM HEPES and mixed (5:1) with 1 mM DTT solution in a 96-well plate. The plate was
covered and incubated for 60 minutes at 37 °C, then centrifuged at 4 °C and 3000 g for 15 minutes to remove
NPs from the solution. 90 µL of supernatants are mixed with 90 µL of 5 mM of 5,5 '-dithiobis-dithiobis (2nitrobenzoic acid (DTNB) solution (Sigma-Aldrich) and production of NTB (5-mercapto-2-nitrobenzoïc acid)
determined by measuring OD at 405 nm by a spectrophotometer (Multiskan-EX, Thermo Scientific). H2O2 at
500 µM was used as positive control and DTT at 200 mM without addition of DTNB as a negative control. A
control for NP absorbance at 405 nm was included, where DTT solution was replaced by 15 mM HEPES. A
blank of 2.5 mM DTNB was also performed. Results are expressed as consumption of DTT in percentage of
control (200 mM DTT in 15 mM HEPES) using the standard curve obtained by incubating 0-250 µM DTT.
Antioxidant depletion assay
The depletion of the antioxidants in an artificial respiratory tract lining fluid (RTLF) consisting of
reduced glutathione (GSH), oxidized glutathione (GSSG), uric acid (UA) and ascorbic acid (AA) was measured
by HPLC (Mudway et al. 2001; Ayres et al. 2008).
Briefly, NPs were incubated for 4 hours at 37 °C in RTLF containing physiological concentrations
(200µM) of UA, AA and GSH at pH 7. Samples were filtered in 0.22 µm spins (Costar, VWR, Fontenay-sousbois, France) and centrifuged for 60 seconds at 12000 g to eliminated NPs from solution. Final concentrations of
antioxidants were quantified by reverse phase high-pressure liquid chromatography (HPLC, Shimadzu, Noisiel,
France). Samples were injected in C48 column (150 mm length, 4.6 mm internal diameter and 5 µm particle
size). Mobile phase was constituted of a gradient system of 25 mM Sodium Phosphate Monobasic; 0,5 mM
Octane Sulfonic Acid (pH 2.7) for the mobile phase A and 100% acetonitrile for the mobile phase B. The
gradient programme was 0-5 min 0 % B; 5-10 min 0-12 % B; and 100 % A for 10 minutes. Products were
studied by spectrophotometry at 243 nm (AA), 280 nm (UA) and 210 nm (GSH; GSSG) and quantified by
integration of the peak area using LabSolution software.