Detailed Materials and Methods
Nanocomposite copper oxide (CuO) particles (nanocrystallite form with a diameter of 210 nm according to the manufacturer’s data Supplement Table S4) were from
NanoScale Material Inc. (USA). All chemicals and biological materials were purchased
from Sigma Aldrich unless stated otherwise.
PARTICLE CHARACTERISATION: Nanoparticle hydrodynamic diameter and zeta
potential were measured on a Malvern Nanosizer ZS at CuO NP concentration of 0.017
mg/ml. Size and zeta potential measurements were measured over a 6 h period and were
averaged results from 3 runs at 37°C in deionized water, cell culture medium without
serum (CCMFBS-) and cell culture medium with 2% (v/v) fetal bovine serum
(CCMFBS2%) which was used in the study for exposing the cells to CuO NP. Ex vivo
oxidative potential of CuO NP was measured by ascorbic acid depletion assay according
to Kelly et al.1. Briefly, 160 µL NP suspension (3.125 µg/mL) were added to UVtransparent 96-well flat-bottomed plates (Greiner bio-one) and pre-incubated for 10 min
at 37°C in a plate reader (Spectra Max 190). Ascorbic acid solution 1 mM was prepared
in Chelex® water pH 7, 40 µL was added to each well and ascorbic acid absorbance was
recorded every 2 min for a period of 2 h by measuring the absorbance at 265 nm. After
subtracting background absorption from the particles, ascorbic acid concentration (mol)
was plotted against time (s) and the initial rate oxidation was determined by linear
regression at t=0-7200 s. The experiment was performed in triplicate and values
expressed as mean ± sd nM s-1 oxidation of ascorbic acid (Supplement Table S5).
IN VITRO TOXICOLOGY EXPERIMENTS: Human alveolar epithelial cells (A549,
ATCC, USA) were cultured in an oxygen cabinet (Don Whitely Scientific, UK) under
normoxic conditions (13% O2) and in a separate incubator under hyperoxic conditions
(21% O2). Oxygen tension in the medium prior to use was confirmed by OxyMini
Oxygen Meter (WPI Inc., Hertfordshire, UK). The cells were cultured using
CCMFBS10% in a humidified incubator at 37°C and 5% CO2. All experiments were
performed on cells seeded at a density of 30,000 cells/cm2 in CCMFBS2% (Fig 2a). For
all experiments, particles were sterilized by dry heat sterilization at 180°C for 20 min
(Memmert, Schwabach, Germany) and then suspended at 1.7 mg/mL in CCMFBS2%.
The suspension was sonicated for 5 min using a probe sonicator at 40 Hz (Vibra Cell
1
Sonics Material Inc. Danbury, CT, USA) and immediately diluted with CCMFBS2%.
NP surface area doses ranging from 0.002 – 2.0 cm2/cm2 (0.08 - 80 μg/ml) were chosen
based on the findings of Faux and co-workers2 who demonstrated that 1 cm2/cm2 is a
critical threshold dose at which non-specific particle-induced inflammation occurs.
BIOCHEMICAL ASSAYS: Intracellular GSH was measured by an adaptation of the
original method described by Senft et al.3. Briefly, A549 cells were cultured on 6-well
plates and exposed to varying concentration of CuO NP for 6 h after which the
supernatant was removed and the cells washed twice with ice cold PBS before addition
of 6.5% v/v trichloroacetic acid (TCA) for 10 min on ice. The TCA extract was
collected for glutathione (GSH) measurement, mixed with O-phthaldialdehyde (OPA)
and the fluorescence from the GSH-OPA adduct was measured using a Hidex
Chameleon fluorometer (Hidex, Turku, Finland) at a λexcitation of 350 nm and λemission of
420 nm. The cells were then lysed by incubation with 0.5 M sodium hydroxide (NaOH)
for 1 h at room temperature and the NaOH extract was collected for total protein
measurement. Total protein was measured using the bicinchoninic acid method4. The
fluorescence per mg protein was calculated and the results were plotted as nmol
intracellular GSH per mg protein.
ROS generation was assessed over a 4 h NP incubation period. Intracellular
ROS levels were measured using dihydrorhodamine -123 (DHR-123, non-fluorescent), a
redox sensitive dye which enters living cells passively where it is oxidised by ROS
(peroxide and peroxynitrite) to the fluorescent compound rhodamine-123 (R-123),
which is then targeted to mitochondria. R-123 fluorescence was measured in live cells
using an atmosphere-controlled plate reader (BMG Labtech), so as not to alter O2 levels
while making the measurements.
ROS was measured by methods described by
Henderson et al. and Royall et al.5,6, using dihydrorhodamine-123 at a final
concentration of 20 µM (DHR-123 Sigma Aldrich, UK). Live cell fluorescence was
measured using an atmospheric controlled fluorescence plate reader every hour at a
λexcitation of 490 nm and λemission of 530 nm up to 4 h. Total protein was measured in cell
lysates at the end of the experiment using the bicinchoninic acid method4. The
fluorescence per mg protein was calculated and the results were presented as % ROS
generated relative to the medium control for each concentration of CuO NP. To obtain a
2
more robust assessment of the temporal generation of intracellular ROS, an area under
the curve value representing the % ROS generation above baseline over 4 h (unit: % h)
was calculated for each nanoparticle concentration.
The effect of CuO NP on cellular metabolic activity/viability was measured by a
reduction in metabolic activity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay7. It should be noted that Wang et al.8,
showed that NP-induced intracellular superoxide generation was also able to reduce
tetrazolium salts to produce artificially high quantities of formazan end products
measured in the MTT assay. In our study, NP-treated cells cultivated at physiological
alveolar O2 concentration (13%) showed increased levels of ROS (Figure 3), which
likely resulted in artificially high formazan quantities measured in the MTT analysis of
these cells, while the MTT results for cells cultivated at 21% O2 may not have been as
heavily influenced by excess ROS, due to the increased levels of GSH present in those
cells.
qRT-PCR: A549 cells were seeded in 6-well plate at 30,000 cells/cm2, were cultured in
13% O2 and 21% O2 and were treated with medium only (no NP), 8 µg/ml and 40 µg/ml
of CuO NP. After 24 h exposure to NP the supernatant was removed and cells were
washed 2 times with cold (4°C) PBS. Total RNA extraction from cells was performed
using RNeasy® kits (Qiagen) following the manufacturer’s instructions, and quantified
with a Nanodrop® 1000 (Thermo Scientific). The High Capacity RNA-to-cDNA reverse
transcription kit (Applied Biosystems) was employed for reverse transcription of RNA
into cDNA. Q-RT-PCR was carried out using the Applied Biosystems 7900HT Fast
PCR system with TaqMan® probes for specific genes of interest. The housekeeping
genes used for cycle thresholds normalization were Beta Cytoskeletal Actin (ACTB),
Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and Polymerase (DNADirected), Delta Interacting Protein 3 (POLDIP3). However, only POLDIP3 was used as
the reference gene for normalization of cycle thresholds as ACTB and GAPDH showed
high variance. A hot start at 95°C for 10 min was followed by 40 cycles at 95°C for 15 s
and 65°C for 1 min. Amplification data were analysed using thermal cycler SDS 2.4 and
DataAssist (V3) software packages.
3
The comparative cycle threshold method (ΔCT)
9
was employed for data
analysis. The CT values for all the genes of interest (GOI) were normalized against the
CT value of the housekeeping gene (HKG) (POLDIP3), i.e. ΔCT (GOI) = CT [GOI] –
CT [HKG]. For the control cells (medium only, no NP treatment) ΔΔCT value of cells
cultured in 13% O2 relative to cells cultured in 21% O2 was calculated, i.e. ΔΔCT (GOI)
= [ΔCT (GOI 13% O2) - ΔCT (GOI 21% O2)]. This was used to calculate fold change
(FC) of control cells cultured in 13% O2 relative to cells cultured in 21% O2 (FC = 2ΔΔCt
). Statistical significance of normalised expression level (ΔCT) GOI 13% O2 relative
to that of GOI 21% O2 was considered for P < 0.05 using a paired, two tailed Student’s
t-test.
For the NP-treated cells (0.02 and 1 cm2/cm2) ΔΔCT value of cells cultured
under each O2 condition was calculated relative to their respective control, i.e. ΔΔCT
(GOI) = [ΔCT (GOI NP treatment) - ΔCT (GOI control)]. This was used to calculate FC
of NP treated cells relative to control cells for each O2 condition (FC = 2- ΔΔCt). This FC
of each GOI from cells cultured in 13% O2 was plotted as percentage of FC of each GOI
from cells cultured in 21% O2, i.e. % FC in GOI relative to 21% = (FC GOI 13% O2 /FC
GOI 21% O2) X 100. Statistical significance of FC GOI NP treatment relative to FC in
GOI control was considered for P < 0.05 using a paired, two tailed Student’s t-test.
VOLCANO PLOT: The volcano score which takes into account both the magnitude of
transcript regulation (FC) and the statistical significance of the change (p-value) was
calculated for each GOI cultured under the two O2 conditions using the FC and p-values
calculated above for NP treatment relative to their respective control. The volcano score
is defined as v = log (FC) X log (p-value) and transcripts were considered potentially
dysregulated after NP treatment when v ≤ -0.103 when FC = 1.2 and p = 0.05. The
volcano plot was prepared according to the method of Glaves et. al.10.
TEM AND EDX-HAADF TEM: A549 cells were grown to confluence on 13 mm
diameter glass coverslips. For TEM, cells on coverslips were fixed by replacing the
medium with 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer and incubation for 2
h at 4°C. After fixation the coverslips were rinsed several times with 0.1 M phosphate
buffer followed by treatment with 1% (w/v) osmium tetroxide in 0.1 M phosphate buffer
(pH 7.3) for 20 min at 4°C. The coverslips were rinsed (10 min) in phosphate buffer and
4
dehydrated in a graded series of ethanol 0 to 100%. The coverslips were then flooded
with TAAB epoxy resin and left to infiltrate for 4 hours at room temperature. To section
in the plane of the monolayers, the coverslips were embedded by inverting them
(monolayer side down) onto a capsule (TAAB) overfilled with resin and then
polymerized for 24 h at 70°C. Ultrathin sections (70-90 nm) were prepared using a
Reichert-Jung Ultracut E ultramicrotome, mounted on 150 mesh copper grids,
contrasted using uranyl acetate and lead citrate and examined on a FEI Tecnai 12
transmission microscope operated at 120 kV. Images were acquired with an AMT
16000M digital camera. At least 6 images from each condition were acquired and
analysed using ImageJ 1.47 software. Features of cellular apoptosis were assessed by
visual inspection of the 6 representative TEM images, based on the presence of nuclear
condensation (pyknosis) and fragmentation (karyorrhexis), the loss of the nuclesomes,
the presence of apoptotic bodies and evidence of crescent shaped condensed chromatin
lining the nuclear membrane. Autophagic vacuoles were identified as double
membranous vacuoles containing morphologically intact cytoplasmic materials11.
DATA ANALYSIS: A Student’s t-test and one-way ANOVA were used to perform the
statistical analysis. Graphical and statistical analysis was performed by using GraphPad
Prism 5 software and IBM SPSS Statistic 20.
5
Supplementary Results
Figure S1 Representative TEM images of A549 cells cultured at 13% (top panel) and
21% (bottom panel) O2.
6
Figure S2 Figures to illustrate the effect of different culture conditions on ROS
generation in A549 cells over 4 h exposure to CuO nanoparticles surface area dose (A to
E; 0.0002 to 2 cm2/cm2). The data represent duplicate experiments of n=3 and this was
used to calculate the area under curve to obtain respective AUC values (% h) of ROS
generation in cells cultured under atmospheric vs. physiological oxygen levels.
7
Figure S3 Effect of hydrogen peroxide and diethyl maleate on ROS generation and
glutathione depletion respectively. Top panel – A549 cells were treated with 2.5 µM
hydrogen peroxide for 4 h and intracellular ROS production was measured using an
atmosphere-controlled plate reader (BMG Labtech). Bottom panel – A549 cells were
treated with 100 µM diethyl maleate for 2 h and GSH concentration was measured in the
lysates. The data represent duplicate experiments of n=3.
8
Table S1: Review of studies comparing the impact of culturing cells under atmospheric and
physiologic oxygen tensions.
Author
Reference
Key findings
12
THP-1 cells were culture at 5% O2 and 18% O2 and the authors
found an increase in metabolic activity at 5% compared to 18%,
significantly decreased phagocytic activity of differentiated THP-1
cells and significant attenuation of NFκB activation.
Neuroblastic cells (SH-SY5Y) cultured at 2-5% oxygen exhibited
low ROS production mimicking levels in primary human cortical
neurons. SH-SY5Y cells cultured at 5% oxygen also exhibited a
greater sensitivity to rotenone than those cultured at atmospheric
oxygen concentrations.
Human nucleus pulposus cells grown 5% oxygen had reduced ROS
generation compared with those cultured under atmospheric oxygen.
Culturing mouse primary mesenchymal stem cells at 5% oxygen
reduced ROS production, increased cell viability and promoted cell
proliferation relative to cells maintained at atmospheric oxygen.
Gene expression patterns in primary glioma cultures at 5% oxygen
closely paralleled those seen in patient tumors in situ. Culturing these
cells at 21% resulted in a drift from the physiologic gene pattern,
which could be restored by cell transfer back to 5% oxygen.
Primary rat neurons cultured at physiological oxygen (5%)
demonstrated higher polarization, lower ROS production, and altered
mitochondrial morphology. When these cells were challenged with
HIV virotoxin increased cell death was observed, whilst cells
maintained at 21% oxygen became hyperpolarized with no increase
in cell death.
Mouse primary hepatocytes were cultured at 10% and 21% oxygen
and challenged with acetaminophen for 6 h and 15 h. The authors
found that the cells cultured at 21% showed an increase of the
mitochondrial oxidant stress, the subsequent decline of the
mitochondrial function, and cell death appeared to be accelerated and
more amplified under 21% oxygen.
Culturing human embryonic stem cells at 5% oxygen induced cell
proliferation and pluripotency compared with cells maintained at
atmospheric oxygen.
Human embryos and oocytes maintained at 5% oxygen throughout
the pre-implantation period demonstrated an increased blastulation
rate, with a higher proportion of optimal embryos for subsequent
implantation, compared with those incubated at atmospheric oxygen.
Culturing primary T cells at 21% oxygen decreased intracellular
glutathione levels compared with cells at 5%, indicative of increased
cellular oxidative stress. Cells cultured at 21% displayed increased
rates of proliferation.
Culturing mouse embryos at 5% oxygen resulted in a global gene
expression pattern that closely resembled that seen in vivo control
embryos.
Murine zygotes at 5% oxygen showed enhanced embryo
development compared with those cultured under atmospheric
Grodzki, A. C. et al.,
2013
Villeneuve, L, et al.,
2013
Nasto, L.A, et al.,
2013
Boregowda , S.V et
al., 2012
13
14
15
Olin, M.R et al.,
2011
16
Tiede, L.M et al.,
2011
17
18
Yan, H.M. et al, 2010
Forristal, C.E et al.,
2010
Kovacic, B &
Vlaisavljevic, V,
2008
Atkuri, K.R et al.,
2007
Rinaudo, P.F, et al.,
2006
Orsi, N.M and Leese,
H.Y, 2001
19
20
21
22
23
9
Zhou, L, et al., 2000
24
Alaluf. S et al., 2000
25
Sato, A et al., 2000
26
oxygen.
Atmospheric oxygen inhibited proliferation of human microvacsular
endothelial cells and reduced synthesis of a range of proteins (Type
IV collagen, platelet endothelial cell adhesion molecule, von
Willebrand factor) compared to cells maintained at 5% oxygen.
Atmospheric oxygen induced human dermal fibroblasts to switch
from a mitotic to a post-mitotic phenotype; a response which was
largely absent in cells cultured under physiological oxygen (4%).
This effect could be impaired by addition of antioxidants, suggesting
oxidative stress was involved in the cellular transition.
Mouse peritoneal macrophages were cultured at 2% and 20 %
oxygen and challenged with LPS. The authors showed that both LPS
and oxygen strongly induce the expression of xCT mRNA without
significantly altering γ-GCS mRNA levels. This increased the
cystine transport activity resulting in increased intracellular GSH
level.
10
Table S2: Details of the genes selected to examine the hierarchical response of airways cells to oxidative stress, together with previous
reports that they are responsive to nanoparticulate challenge. .
Genes
GCLC
GSTK1
HMOX1
KEAP1
Adaptation
PLAA
MT2A
SOD1
CYP1A1
GPX1
CAT
NQO1
CHUK
Inflammation
Full name
Glutamate—cysteine ligase
catalytic subunit
Glutathione S-transferase
kappa 1
Heme Oxygenase 1
Kelch-like ECH-associated
protein 1
Phospholipase A2-activating
protein
Metallothionein 2A
Cu,Zn Superoxide Dismutase
Cytochrome P450, family 1,
subfamily A, polypeptide 1
Cellular Glutathione
Peroxidase
Catalase
NAD(P)H:Quinone
Oxidoreductase 1
Conserved Helix-Loop-Helix
Ubiquitous Kinase
Interleukin-8
IL8
MIF
NOS2
RefSeq#
Function
NM_001498
Glutathione synthesis
NM_015917
NM_002133
Cellular detoxification
Haeme catabolism
Nanoparticle
Responsive
27, 28
29
X
28, 30, 31
28, 32
X
NM_012289
Nrf2 regulation
Possible tumor
NM_001031689 suppressor
NM_005953
Metal chelation
NM_000454
Antioxidant defense
Xenobiotic
NM_000499
metabolism
NM_000581
NM_001752
NM_000903
NM_001278
NM_000584
Macrophage Migration
Inhibitory Factor
Inducible Nitric Oxide
Synthase
Regulatory elementsa
Nrf2 AP1 NfkB P53
X
X
NM_002415
NM_000625
Antioxidant defense
Antioxidant defense
Xenobiotic
metabolism
NFκB regulation
Inflammatory
chemokine
Pro-inflammatory
cytokine
Induced nitric oxide
production.
X
X
X
X
X
33
32, 33
34
X
35
X
X
36
28
X
37, 38
X
X
31
X
X
39
X
X
40
11
IL6
TNF
LTA
Interleukin-6
Tumor Necrosis Factor
Lymphotoxin Alpha
Ataxia Telangiectasia Mutated
ATM
Cell cycle
arrest and
cell death
Growth Arrest And DNAGADD45B Damage-Inducible, Beta
Annexin A5
ANXA5
Caspase 1
CASP1
X-Ray Repair CrossXRCC1
Complementing Protein 1
Checkpoint kinase 2
CHEK2
Mouse Double Minute 2,
Human Homolog Of; P53MDM2
Binding Protein
NM_000600
NM_000594
NM_000595
Inflammatory cytokine
Inflammatory cytokine
NM_001154
NM_033292
Activation of DNA
repair
Cell growth and
apoptosis
Exocytosis and
endocytotosis.
Apoptosis
NM_006297
NM_007194
DNA repair
Cell cycle arrest
NM_000051
NM_015675
X
X
X
X
X
X
X
X
X
X
31, 41
41, 42
41
43
X
33
X
44
45
X
X
NM_002392
46
47
Regulation of p53
The presence of transcription binding sites for Nrf2, AP-1, NFkB and p53 (X) within a region 20kB upstream of the gene of interest was
assessed using the SABiosciences text mining application and the UCSC genome browser:
http://www.sabiosciences.com/chipqpcrsearch.php?app=TFBS. In addition a detailed search of the literature was conducted for the
presence of antioxidant response elements (ARE), 12-O-Tetradecanoylphorbol-13-acetate (TPA) response elements, kB sites and p53
response elements upstream of the genes, as well as evidence of induction by known inducers of the various transcription factors.
12
Table S3: The statistical significance for the change in gene expression between cells
cultured under 13% and 21% oxygen following CuO nanoparticle treatment compared
against their respective medium controls (no NP treatment). Baseline comparisons in
unchallenged cells cultures under the two oxygen concentrations are also illustrated.
Statistical analysis was carried out using Student’s t-test (paired, two tailed). # P<0.10, *
P<0.05, ** P<0.01, *** P<0.001, ns = not significantly different.
13
Table S4 Properties of CuO NP as supplied by manufacturer.
Properties
NanoActive® Copper Oxide
Appearance/Color
Black Powder
Specific Surface Area(BET) (m2/g)
≥ 65
Crystallite Size (nm)
≤8
Average Pore Diameter (Å)
85
Total Pore Volume (cc/g)
≥ 0.1
Bulk Density (g/cc)
1.65
True Density (g/cc)
5.7
Mean Aggregate Size, d0.5 (μm)
6
Moisture Content (%)
≤4
Loss on Ignition (%)
≤4
Content (Based on Metal) (%)
> 99.6
Table S5: Particle size of copper oxide nanoparticles in water or cell culture medium containing
no fetal bovine serum (FBS) or at concentrations of 2% v/v (CCM FBS2) over a period of 6 h. The
size was measured at 37°C every 30 minutes. Oxidative potential was measured by ascorbic acid
depletion. Data represent the mean ± SD; n=3.
Material Suspension Medium
CuO
Zeta Potential Oxidative Potential
(mv)
nmol/second
Particle Size (nm)
0h
6h
Deionized water
-31 ± 3
11 ± 1
852 ± 291
2264 ± 2263
CCMFBS-
-
-
783 ± 41
7203 ±4071
CCMFBS2%
-11 ± 1
-
283 ± 23
251 ± 3
14
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