Toxicity evaluation of manufactured CeO2 nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells
Toxicity evaluation of manufactured CeO 2 nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells
Background: Engineered nanomaterials may release nanosized residues, by degradation, throughout their life cycle. These residues may be a threat for living organisms. They may be ingested by humans through food and water. Although the toxicity of pristine CeO2 nanoparticles (NPs) has been documented, there is a lack of studies on manufactured nanoparticles, which are often surface modified. Here, we investigated the potential adverse effects of CeO2 Nanobyk 3810 NPs, used in wood care, and their residues, altered by light or acid. Results: Human intestinal Caco-2 cells were exposed to residues degraded by daylight or in a medium simulating gastric acidity. Size and zeta potential were determined by dynamic light scattering. The surface structure and redox state of cerium were analyzed by transmission electronic microscopy (TEM) and X-ray absorption spectroscopy, respectively. Viability tests were performed in Caco-2 cells exposed to NPs. Cell morphology was imaged with scanning electronic microscopy. Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions. No change in the cerium redox state was observed for altered NPs. All CeO2 NPs suspended in the culture medium became microsized. Cytotoxicity tests showed no toxicity after Caco-2 cell exposure to these various NPs up to 170 g/mL (24 h and 72 h). Nevertheless, a more-sensitive whole-gene-expression study, based on a pathway-driven analysis, highlighted a modification of metabolic activity, especially mitochondrial function, by altered Nanobyk 3810. The down-regulation of key genes of this pathway was validated by qRT-PCR. Conversely, Nanobyk 3810 coated with ammonium citrate did not display any adverse effect at the same concentration. Conclusion: The degraded nanoparticles were more toxic than their coated counterparts. Desorption of the outside layer was the most likely cause of this discrepancy in toxicity. It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.
Engineered nanomaterials; Nanoparticles; Transcriptome; Toxicogenomics; Life cycle
The use of nanoparticles (NPs) has increased
significantly during the last decade in several areas such as
computer science, chemistry, cosmetics and
pharmaceuticals. There is an urgent need to verify their
harmlessness in relation to human health and the environment.
Cerium oxide (CeO2) NPs are one of the most widely
used types, for UV protection in paints or as fuel
additives [1,2]. These NPs are usually surface modified to be
incorporated into the final commercialized products.
Contrary to toxicity studies regarding pristine CeO2
NPs, very few studies have focused on manufactured
nanoparticles, which are often surface treated to improve
their dispersion in liquids. In recent years, CeO2 NPs
have been shown to induce loss of viability  and
apoptosis  in human lung cells through ROS production,
as well as DNA and chromosome damage to human
dermal cells . However, these results are controversial.
Xia et al. showed a lack of toxicity of CeO2 NPs and a
protective effect against exogenous ROS . This result
is in agreement with the neuroprotective effect of CeO2
nanoparticles observed by Schubert et al. . So far, a
clear answer to the question as to whether engineered
CeO2 nanoparticles are toxic or cross biological barriers
cannot be provided and more work is required. In
addition, these studies focused mainly on inhalation
rather than oral exposure, which is also a potential route.
To our knowledge, the toxicity of CeO2 NPs to intestinal
cells was evaluated only recently by B. Gaiser et al. .
The authors suggested that both micro- and nanosized
CeO2 NPs can be taken up by Caco-2 cells, but with
little biological consequence, although they suggested that
further work would be required to investigate this in
more detail. All of these studies focused on the toxicity
of pristine CeO2 NPs, i.e. at the beginning of the NPs
chain value. However, the NPs that spread in the
environment are likely to be degradation residues of
CeO2based nanomaterials. Exposure to the environment (UV,
water and air contact ) may alter the physicochemical
properties of surface-modified CeO2 NPs, such as the
chemical structure of the surface, size, shape, and
dispersion state, which are important parameters for toxicity.
After ingestion, stomach acidity can also alter the
physicochemical properties of surface-modified CeO2 NPs,
and their toxic properties. For instance, Wang et al.
described an increase in CdSe NP toxicity in intestinal cells
after acid treatment, due to degradation of the PEG
protective layer .
Our study focuses on CeO2-based nanomaterials at
several stages of the product life cycle. CeO2 NPs usually
refer to uncoated NPs with UV filter properties.
However, CeO2 NPs are often formulated prior to use as
outdoor paint adjuvants, i.e. the commercialized product,
Nanobyk 3810 (named NB in the text), from the Byk
Company. This Nanobyk formulation comprises a core
of CeO2 NPs with a triammonium citrate coating that
improves their dispersion in water and paint [10,11]. The
question arises as to whether, during its life cycle,
especially by exposure to daylight, this material transforms
into a more toxic form for living organisms and the
This work aims to evaluate the relative toxicity of
manufactured CeO2 Nanobyk NPs (NB) compared with
their degraded counterparts. Two alteration protocols
were used. Firstly, extreme and long-term environmental
conditions of aging (100% hygrometry and permanent
sunshine) were reproduced, leading to a light-degraded residue
(NB-DL). Secondly, gastric degradation was mimicked using
a simulated gastric medium, to generate an acid-degraded
residue (NB-DA). Surface-untreated (pristine) CeO2
NPs from Rhodia were used as a comparative material.
Physicochemical properties (e.g., shape, size,
aggregation state, zeta potential and crystal structure) were
determined using dynamic light scattering (DLS) coupled
to laser Doppler microelectrophoresis for zeta potential
measurement, and transmission electron microscopy
(TEM). The surface structure and redox state of
cerium (Ce4+ versus Ce3+) were analyzed by X-ray
absorption spectroscopy (XAS) [5,12].
We used the Caco-2 cell line as a human intestinal
epithelium model. This cell line has been extensively
characterized and shown to exhibit a faithful
representation of in vivo structural characteristics. At the
molecular level, these cells mirror the differentiation of human
intestinal cells [13,14].
We used two viability tests to determine toxic
concentrations of these nanoparticles in Caco-2 cells (ATP
intracellular measurement and XTT test). Several tests
based on different principles are often necessary because
NPs may sometimes interact with the test principle
[15,16]. The first cytotoxicity test is one of the most
sensitive toxicity assays because it is based on the
measurement of ATP, which reflects the energy state of the cell,
even before any damage to membrane integrity occurs.
The second, and more usual, test (XTT) is based on the
activity of mitochondrial enzymes. These tests are
routine tests attesting to the presence of dead cells.
Nevertheless, some deleterious effects may occur before cell
death (inflammation, sensitization, oxidative stress). This
is why we also used toxicogenomics, meaning analysis of
the whole genomic expression with human pangenomic
microarrays to obtain an overview of intracellular events
triggered by these various NPs. Additionally, using the
same methodology, we also examined the adverse effects
elicited by pristine CeO2 NPs as comparative material.
Hydrogen peroxide was used as positive control, as its
effect in Caco-2 cells has been described . We
compared the expression profiles of cells exposed to these
various particles before and after alteration, using a low
concentration (20 g/mL) about eight times lower than
that producing the first visible loss of viability by XTT
test. We used scanning electronic microscopy (SEM) to
visualize potential adsorption of aggregates onto the cell
surface. This multipronged approach gives more certainty
and coherence to the acquired data.
Physicochemical behavior of (un)altered Nanobyk NPs
and pristine CeO2 NPs
Unaltered Nanobyk NPs and pristine CeO2 NPs were
characterized in pure water. In both cases, TEM showed
well-crystallized clusters of cerianite with an inter-reticular
distance (dhkl) measured at around ~3.2 (close to the
d111 of CeO2). These clusters were pseudospherical with
diameters of 3 1 nm (average of 50 measurements)
(Figure 1). In the stock suspension, the pristine CeO2
NPs and unaltered Nanobyk NPs were colloidally stable,
with average hydrodynamic diameters (Dh) of ~7 nm. At
pH ~ 7 0.1, the zeta potential of the Nanobyk NPs was
negative (45 5 mV) compared with the positive zeta
potential of the pristine CeO2 NPs (28 2 mV). After both
environmental (light) and gastric (acidic) degradation, the
zeta potential of the Nanobyk NPs increased (28 2 mV
and 19 2 mV, respectively, at pH 7 0.2). No change in
the Ce redox state was observed between the unaltered
Nanobyk, Nanobyk DL and Nanobyk DA (Additional
file 1: Figure S1).
The dispersion of NPs has a crucial impact on toxicity.
We used DLS to measure the apparent hydrodynamic
diameters of NPs in different media. In water, the
pristine CeO2 NPs and unaltered Nanobyk NPs were
dispersed and stable with average hydrodynamic diameters
(Dh) of 7 nm. In culture medium, with or without
serum, they formed aggregates above 1 m size.
Lightand acid-degraded NPs formed similar aggregates
whatever the medium (water, serum-free medium and medium
supplemented with 10% FCS). In the presence of 10%
serum, in our hands, DLS analyses were especially
unreliable, whatever the NPs and concentrations, giving average
sizes approaching 2 m with particle dispersion indexes
Figure 1 High-Resolution Transmission Electron Microscope images of CeO2 NPs and Nanobyk NPs in deionized water. A) uncoated
CeO2 NPs, B) NB NPs before treatment, C) NB NPs after 4 months aging in daylight (NB-DL), and D) NB NPs after acidic degradation (NB-DA). The
inter-reticular distance (dhkl) measured at ~3.2 is attributed to the (111) crystalline plane of CeO2. No changes in shape, crystal structure, or
CeO2 cluster size were observed by HRTEM.
close to 1. In serum-free medium, the mean hydrodynamic
diameters were 1580 1000 nm, 1030 370 nm, 1300
190 nm, and 2200 500 nm for the NB, NB-DL, NB-DA,
and the pristine CeO2, respectively. It is noteworthy that the
samples were not sonicated before any of the toxicity assays.
The studies were conducted on the well-established
Caco-2 cell line, differentiated for 21 days. The integrity
of the cell layer was assessed by measurement of the
transepithelial electrical resistance (TEER), stabilized at
500 ohms.cm2 after 21 days. We performed two
cytotoxicity tests at two exposure times, 24 h and 72 h, to
take into account the kinetic parameters. As shown in
Figure 2, with the ATP assay (Left side), the presence
of CeO2 NPs did not induce adverse effects on
Caco2 cells after 24 h or 72 h exposure, even at high
concentration (170 g/mL). The XTT assay (Right side)
confirmed the lack of apparent toxicity of Nanobyk NPs and
pristine cerium NPs in Caco-2 cells. For degraded
Nanobyk NPs (NB-DL and NB-DA) only mild toxicity was
observed using the XTT test, and only at the highest
concentration (170 g/mL) after 72 h exposure.
Cell morphology after exposure
We visualized cells with SEM. Caco-2 cells exposed for
72 h to 170 g/mL Nanobyk-type NPs did not show any
alteration in density. The microvilli were also clearly
visible (Additional file 2: Figure S2). These observations
confirmed the absence of visible toxicity of Nanobyk
NPs and indicated that, if an effect exists owing to
exposure to degraded NB, this effect is small enough not
to induce any drastic changes in cell morphology. It is
noteworthy that this does not exclude any possible
metabolic modification. Nevertheless, clear spots identified as
CeO2 deposits were clearly visible on the surface of the
plasma membrane, especially for NB-DL (Figure 3).
Caco-2 cells were exposed to Nanobyk, Nanobyk DL,
Nanobyk DA and pristine CeO2 for 72 h at 21.25 g/
mL. Cells were also exposed to hydrogen peroxide as a
positive control. Agilent Oligo microarrays spotted with
4 x 44,000 probes were hybridized in quadruplicates
with RNAs originating from cells exposed to NPs, or from
unexposed cells (for exact design details, see Methods).
Additional file 3: Figure S3 represents the scatter plots of
Figure 2 Caco-2 cell viability tests. Caco-2 cells were grown in 96-well plates and differentiated for 21 days. Cells were then exposed for 24 h
or 72 h to concentrations of CeO2 NPs ranging from 21.25 to 170 g/mL. Left side) ATP tests: cell viability was determined by reading the level
of bioluminescence (CellTiter-Glo luminescent cell viability assay, Promega). Right side) XTT tests: cell viability was determined by mitochondrial
enzyme activity via XTT reagent (In Vitro toxicology assay kit XTT based, Sigma-Aldrich). An experimental positive control was obtained by
exposing cells to H2O2 in both tests. Cell viability was not altered for concentrations up to 170 mg/L.
Figure 3 SEM image and cerium characterization. Left side: SEM image, obtained in BSE mode, of Caco-2 cells exposed to Nanobyk-DL.
Magnification 500 x. Right side: The EDX spectrum of the clear spots detected on the cell membrane indicates they are composed of cerium.
the raw fluorescence intensities of genes filtered on the
threshold intensity signal of the microarray experiments
(n = 4). The scatter plot obtained for Nanobyk NPs versus
control 1 was very similar to the control scatter plot
representing the raw intensities of genes obtained from
untreated cells from two different cultures (control 2 versus
control 1) giving the false positive rate (5 genes). The
exact number of significantly altered genes (fold change
1.5, p-value <0.05) in each probe set is reported in Table 1.
The exhaustive list with gene names and fold changes is
shown in Additional file 4: Table S1 under Supporting
Information (see Additional file 4).
Cells exposed to NB showed almost no change in their
gene expression, only 13 genes having significantly
modified expression. Conversely, cells exposed to degraded
NPs had a different scatter plot from the control scatter
plot, and had 344 and 428 modified genes for NB-DL and
NB-DA, respectively, with 37 common genes listed in
Additional file 5: Table S2 under Supporting Information,
Table 1 Microarray results
(see Additional file 5). Cells exposed to pristine
(surfaceuntreated) CeO2 NPs displayed much stronger
deregulation of their gene expression, with 1643 modified genes.
Furthermore, a positive-control scatter plot was obtained
with cells exposed to hydrogen peroxide for 24 h at
20 M (9307 modified genes).
Biological analysis of the transcriptome
The lists of altered genes were then processed using
Ingenuity Pathways Analysis to investigate a possible
relationship between altered genes and mechanisms of
For pristine CeO2 NPs, the main altered functions
were cellular growth and proliferation (274 genes) and
cell death (265 genes), as shown in Figure 4. Contrarily
to pristine CeO2 NPs, the surface-treated NB NPs did
not alter any specific cellular function (not shown in
Figure 4, as only 13 genes are altered), thus proving
Caco-2 cells were cultured and differentiated for 21 days. The cells were exposed for 72 h to 21.25 g/mL CeO2 NPs, surface-treated or degraded (n = 2). Pristine
CeO2 NPs at the same concentration and H2O2 (20 M) were used as positive controls. After mRNA extraction, labeled cDNA (Cy3) was hybridized (n = 4) to an
Agilent oligomicroarray (4 44,000 probes). The number of genes detected above the signal threshold was compared for each type of NP versus their own
control. From these remaining spots, we selected those with fluorescence ratios (representing NP-treated samples versus untreated samples) above 1.5-fold change. Out
of these spots, we selected those satisfying Benjamini-Hochberg multiple testing corrections. At the end of this analysis, we obtained lists of genes that were significantly
induced or repressed after exposure to NPs.
Figure 4 Radar plots of gene distribution per altered function. Genes significantly induced or repressed after exposure to NPs were selected
as described in Table 1, Column 4. Genes were selected with fold-change ratios greater than 1.5 (n = 4, p-value 0.05), and distributed per
function. This graph displays the number of significantly altered genes per function. The enlarged part concerns light-degraded Nanobyk
(NB-DL) and acid-degraded Nanobyk (NB-DA). For each compound, a pattern is obtained representing the amplitude and the nature of its
toxicity, then allowing a visual comparison of their respective toxicities.
the efficiency of the protective triammonium citrate
layer. The NB nanoparticle residues of degradation by
light or acid altered similar functions to pristine CeO2
NPs, although in a very moderate way, given the
number of altered genes. The distribution per function for
NB residues is shown in the enlarged part of Figure 4.
For NB-DL, the main altered functions were cellular
growth and proliferation (51 genes), cellular
development (35 genes), small molecule biochemistry (26 genes),
cellular assembly and organization (25 genes), cell cycle
(24 genes), cellular function and maintenance (24 genes)
and cell death (20 genes). Other functions counted for less
than 15 genes each.
For NB-DA, the altered functions were molecular
transport (53 genes), small molecule biochemistry (45 genes),
cellular assembly and organization (32 genes), nucleic acid
metabolism (23 genes), cellular function and maintenance,
lipid metabolism and protein synthesis (22 genes each)
and cell death (21 genes). Other functions counted for less
than 15 genes each.
Canonical pathways analysis
If we look more precisely at the main common
molecular pathways definitely impacted by these different NPs
(Figure 5), pristine CeO2 NPs induced mitochondrial
dysfunction through the underexpression of 27 genes of
complexes I, III, IV and V, listed in Table 2 Part A.
Interestingly, the NB-DA residue also induced mitochondrial
dysfunction by underexpressing 10 genes of the
complexes I, II, III, IV and VI, listed in Table 2 Part B.
Nevertheless, in the case of NB-DA this pathway was
affected to a lesser extent. The same trend was observed
with NB-DL, which downregulated 3 genes in this
pathway, reported in Table 2 Part C.
qRT-PCR validation of the microarray data focused on
Validation of the microarray data was confirmed by
quantitative RT-PCR on 8 genes belonging to complexes
of mitochondrial respiratory chain. The compared fold
changes obtained with microarray and with qRT-PCR
were reported in Table 3. All mRNAs were
downregulated with both techniques, with a good level of
significance (p < 0.05).
Contrary to the toxicity studies regarding pristine CeO2
NPs, very few studies have focused on engineered
nanoparticles, which are often surface treated for better
dispersion in liquid products. However, these particles can
Figure 5 Comparative analysis of significantly altered pathways by CeO2 NPs (dark blue), NB-DL NPs (medium blue) and NB-DA NPs
(light blue). The y-axis depicts genes ratio within a dataset mapping to the considered pathway (see Methods for calculation). A fishers exact test
was used to determine a p-value representing the significance of these associations (p < 0.01).
be degraded in the environment during their life cycle.
Moreover, although the toxicity of pristine nanoparticles
via inhalation is well documented [3,18,19], only a few
evaluations of toxicity associated with oral exposure
have been carried out . Here, we investigated the
potential toxic effects on the intestine of Nanobyk 3810
NPs, used as long-term UV protection for wood, and
their degradation residues, as compared with pristine
CeO2 NPs. We considered two degradation scenarios: i)
due to environmental conditions, and ii) due to stomach
acidity. Caco-2 cells were used for toxicity evaluations
and their differentiation was evaluated with the
measurement of transepithelial resistance.
Following both environmental or acidic degradation in
water, the NB surface becomes less negatively charged at
physiological pH than the initial NB (zeta potential 28
2 mV and 19 2 mV for NB-DL and NB-DA,
respectively, compared to 45 mV for NB). These negative
surface charges are attributed to the negatively charged
citrate capping at this pH. Indeed, citrate is a tricarboxylic
acid with three dissociated protons (pKa1 = 3.13, pKa2 =
4.76 and pKa3 = 6.40) . Consequently, partial
desorption of the citrate layer during alteration will decrease the
negative charges at the surface of the Nanobyk, as already
described . While the Nanobyk and the pristine CeO2
NPs have hydrodynamic diameters of 7 1 nm in a
dispersed state, strong aggregation occurs in the culture
medium, with and without 10% FCS. Serum proteins
usually help the dispersal of the NPs , but for very
small NPs with hydrodynamic diameters less than 10 nm
the bonds to proteins are so weak and labile that most of
the NPs exchange quickly, which favors aggregation, as
has been described recently in our laboratory by Liu et al.
. Using a culture medium supplemented with 10%
FCS and containing 60 g/mL CeO2 NPs, NP distribution
with the protein population was analyzed by size exclusion
chromatography and ICP-MS, which showed that 90% of
the CeO2 NP population was eluted in the dead volume as
homo aggregates. This is consistent with the results
obtained by SEM showing adsorbed NP aggregates on the
cell membrane. The hydrodynamic diameters of all NPs
tested, including NB-DL and NB-DA, in the cell culture
medium are microsized, between 1 and 2 m, with zeta
potentials around 15 mV, leading to strong aggregation,
and coherent with published data [18,23,24].
Through two different toxicity assays (ATP and XTT),
Caco-2 showed no loss of viability after exposure to up
to 170 g/mL over 72 h for Nanobyk 3810 and pristine
CeO2 NPs. For degraded Nanobyk NPs, a slight effect
(27% loss of viability) was observed for the highest
concentration (170 g/mL) after 72 h with the XTT test,
but not with the ATP tests, for NB-DL and NB-DA.
ATP and XTT tests are not based on the same principle.
XTT is directly based on the enzymatic activity of
succinate dehydrogenase, essential to mitochondria. We showed
that this enzyme was down regulated in our dataset. ATP
test measures the total amount of intracellular ATP that is
not decreased in a so sensitive way. This explains why the
sole XTT test is disturbed by those NPs. This last test
tends to indicate that altered Nanobyk NPs induce a slight
change in mitochondrial function. It should be also noted
that the XTT test, by induction of a soluble formazan
species, does not suffer from the possible interference with
NPs frequently described with the MTT assay. This
known bias is due to the formation of insoluble MTT
formazan, which is often entwined with NP fibers or
complexes that inhibit its resolution by the solvent
. None of the tested nanoparticles induced drastic
changes in cellular aspect as depicted in Additional
file 2: Figure S2, excepted deposits on cell membrane,
suggesting that the effect was probably a modification
of the internal metabolic activity. It is possible that
some signaling cascades may be triggered by
interaction with membrane proteins such as protein-G
coupled receptors .
We did not notice any evolution of the redox state (i.e.
potential reduction of Ce4+ to Ce3+) of Ce atoms
localized at the surface of the NPs after alteration in water.
The shape of XANES spectra at the Ce L3-edge, and the
position of the edge, are usually easily distinguished for
Ce3+ and Ce4+ reference compounds: one absorption
edge at 5729 eV for Ce3+ and a double peak at 5733 eV
and 5740 eV for Ce4+ (Additional file 1: Figure S1).
Consequently, XANES, used to detect any slight change in
the redox state of CeO2, indicated no Ce3+ in our
Standard toxicological methods in vivo cannot unravel
the mechanisms of action of toxicants. Some in vitro
methods are able to do so, but rarely at sublethal doses.
Conversely, toxicogenomics is a methodology capable of
detecting potential changes in cellular and molecular
functions at low dose, and constitutes an alternative for
assessing the toxicity of nanomaterials . In particular,
microarray-based transcriptional profiling is a powerful
Table 3 Microarray gene expression validation
COX6A2 F:CTACCAACACCTCCGCATC R:TCGAAGCTTCACACCTTTATTG
F = forward; R = reverse.
Table 2 Lists of genes significantly altered by exposure to pristine CeO2 (Part A), NB-DA (Part B) and NB-DL (Part C),
and belonging to the canonical pathway mitochondrial dysfunction (Continued)
succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa
succinate dehydrogenase complex, subunit D, integral membrane protein
Although the same pathway is activated, there are, stricto sensu, no common genes altered by pristine CeO2 and NB-DA or NB-DL in the mitochondrial dysfunction
pathway. This demonstrates the interest of a pathway-driven analysis compared to the usual gene-driven analysis.
Microarray Fold change NB-DL / Ctrl
tool for monitoring altered cellular functions and
pathways (i.e. oxidative stress, apoptosis, hypoxia, etc.) under
the action of toxicants. Gene expression studies, through
a large number of altered genes, provide a wealth of
information for sketching the intracellular mode of action
of toxic substances [28,29]. These results usually lead to
the generation of new hypotheses about the specific
toxicity of the substances concerned, then allowing targeted
in vitro or in vivo experiments. The overall number of
altered genes is a very good indicator of the level of
cellular disturbance induced by a toxic compound.
The cells were exposed to 21.25 g/mL of various NPs
for 72 h, a concentration about eight times lower than
that producing the first visible loss of cell viability at
72 h with the XTT test. This exploratory concentration
was chosen in the absence of any oral reference dose for
cerium oxide nanoparticles. In the current study,
toxicogenomic results showed that, by exposure to NanoByk
NPs capped with ammonium citrate, Caco-2 cells were
almost unaffected, as only 13 genes were altered out of
44,000 transcripts (Table 1). On the contrary, by
exposure to pristine CeO2 nanoparticles used as the reference
material in the same conditions, cells underwent a
strong alteration of gene expression, with 1,643
Microarray Fold change NB-DA / Ctrl
significantly differentially expressed genes. In short,
capping nanoparticles provides a very good protection
against adverse cellular effects. On the other hand,
lightor acid-degraded Nanobyk NPs moderately altered the
transcriptome of Caco-2 cells, with 344 and 428
modified genes, respectively, including 37 common genes
(Additional file 5: Table S2). These alterations were
much less important than those obtained when cells
were exposed to pristine CeO2 NPs at the same
concentration (1,643 genes). For instance, cell death was more
highly activated for CeO2 NPs (265 genes) than for
NBDL and NB-DA (about 20 genes each). Otherwise, we
noted that there was no cytokine production
(chemokines or interleukins), indicating an absence of
proinflammatory response with all the NPs tested.
To identify the mechanisms involved, we analyzed the
distribution of altered genes per function, as defined in
Gene Ontology (Figure 4). Radar plots may help here to
apprehend the complexity of toxicity, both in terms of
amplitude and effect. This results in a specific pattern
representing the toxicity of each compound. Graph
overlay of several products allows the visual comparison of
their respective toxicities. Once again, the magnitude of
the effect is given by the number of altered genes in a
given function. For NB-DL, as for CeO2 NPs, the main
altered functions were similar: cellular growth and
proliferation (51 vs. 274 genes), cellular development (35 vs.
159 genes), cell death (20 vs. 265 genes) and small
molecule biochemistry (26 vs. 140 genes). This may suggest
that their modes of toxic action are similar. For NB-DA,
the main altered functions related to molecular transport
(53 genes) and small molecule biochemistry (45 genes).
The functional mechanisms of toxicity were slightly
different between the two residues, although this type of
analysis is more informative with a higher number of
genes. Small molecule biochemistry was an altered
function in all cases.
Although the functions provide valuable information on
the actions of the involved genes (transport, cell growth,
etc. ..), the pathways help in understanding, in a faster and
more extensive manner, the interactions between these
genes themselves and the cellular mechanisms to which
they belong. In the present study, pristine CeO2 NPs
greatly disturbed the EIF-2 signaling pathway by
downregulating the expression of more than 90 ribosomal proteins
(S and L type), with a strong overall impact on protein
synthesis (Figure 5). This result was consistent with the
results of L. Benameur , where pristine CeO2 NPs
triggered intracellular damage, such as the disruption of
antioxidant systems (GSH, SOD, GPx, ascorbate), and
impacted on protein synthesis in general.
Comparison of the common canonical pathways
disturbed by pristine CeO2 NPs and degraded NB NPs
essentially showed that they all disturbed mitochondrial
functions and the oxidative phosphorylation pathway.
Oxidative phosphorylation is the mitochondrial process
by which ATP is formed as a result of the transfer of
electrons from NADH or FADH2 through a series of
electron carriers to oxygen. Consequently, this pathway is
modified as a result of mitochondrial dysfunction (Figure 6).
The downregulation of some key genes of oxidative
phosphorylation was validated by qRT-PCR (Table 3).
This point confirms the results obtained above with
the XTT test, which involved the mitochondrial
enzymatic process. Moreover, the succinate dehydrogenase
complex is clearly underexpressed by NB-DL and
NBDA (SDHB, SDHC and SDHD) whereas the NADH
dehydrogenase complex (NDUFA, NDUFB and NDUFS) is
downregulated by pristine CeO2 NPs. This impairment
is, however, visible by toxicogenomics at a concentration
divided by 8 (21.25 g/mL) compared to the XTT test
(170 g/mL). This proves that toxicogenomics is more
sensitive, while at the same time being informative about
the molecular actions of toxic substances. Interestingly,
although the same pathway was activated, there were,
stricto sensu, no common genes altered by pristine CeO2
and NB-DA or NB-DL in the mitochondrial dysfunction
pathway as reported in Table 2. Consequently, here, we
demonstrated the interest of a pathway-driven analysis
compared to the usual gene-driven analysis. In other
words, looking at the whole picture is more informative
than looking only at key genes.
In addition, these results were perfectly consistent with
data obtained by D.A. Pelletier at al.,  who
investigated the effects of cerium oxide NPs on bacterial
growth and viability in E. coli, using microarray
technology. These authors highlighted concomitant low levels
of expression of succinate dehydrogenase and
cytochrome b terminal oxidase genes. This indicates that
cerium NPs alter electron flow and the respiratory chain
in mitochondria. P. Rozenkranz et al. also demonstrated
that pristine CeO2 NPs decreased mitochondrial activity
in H4IIE cells .
In the case of the partial or total dissolution of the
triammonium citrate layer of NB, the CeO2 core
becomes exposed. A lack of effect of solubilized
ammonium citrate can be assumed since this compound is
totally metabolized by most cells in the urea
(ammonium) and Krebs (citrate) cycles. Owing to the presence
of oxygen vacancies on its surface, and to the
selfregenerative cycle of its dual oxidation states, CeO2 can
scavenge ROS in biological systems. Indeed, when the
ratio Ce3+/Ce4+ falls, the scavenging capabilities increase
. Through this property, altered NB adsorbed onto
the cell membrane may interact with molecules such as
membrane receptors, as suggested by Lee et al. , or
other molecules, to trigger cell responses via catalytic
properties of bare CeO2. This can only be a low level
Figure 6 Complexes of the respiratory chain altered by degraded Nanobyk. Pristine CeO2 under-expressed 27 genes encoding subunits of
complex I, III ( cytochrome b-c1), IV and V. Acid-degraded Nanobyk downregulated 10 genes encoding subunits of complex I (NADH dehydrogenase),
complex II (succinate dehydrogenase), complex IV (cytochrome c oxidase) and complex V (ATP synthase). Light-degraded Nanobyk down regulated 3
genes encoding subunits of complexes II and IV. Nanobyk NPs did not alter the respiratory chain. Differentially expressed genes belonging to this specific
pathway are listed in Table 2.
response because such interactions likely occur only at
We showed that the properties of residues of Nanobyk
nanoparticles tend to be similar to those of pristine CeO2
nanoparticles, and different from unaltered Nanobyk.
Consequently, cells exposed to light- or acid-degraded
Nanobyk were exposed to nanoparticles having surface
properties that were closer to those of pristine
nanoparticles. The external layer had an efficient protective effect
towards toxicity, as we recently observed elsewhere for
engineered titanium dioxide NPs including an aluminum
oxide protective layer . However, when this layer
was altered, the toxic effects, visible by
toxicogenomics, increased. Acid alteration of nanoparticles was
more severe than light alteration, with regards to the
respiratory chain. Macroscopic measurements revealed
that all of these NPs were aggregated with identical
zeta potential in the culture medium. Thus, the surface
properties are important parameters for the toxicity of
nanoparticles, whatever their aggregation state. This
study examined the acute effects of engineered cerium
oxide nanoparticles, which were very moderate. However,
the question remains as to their long-term effects: this is
another issue to be addressed.
The capping layer of Nanobyk 3810 used in outdoor
paints has an efficient protective effect against toxicity
for intestinal cells. The alteration of this layer, by light or
acidic treatment, causes some toxic effects similar to
those induced by pristine CeO2 nanoparticles. Using
toxicogenomics, we found a modification of cell
metabolism, especially an alteration of mitochondrial function
by underexpression of essential enzymes of the cellular
respiratory chain, caused by these altered nanoparticles.
The modification of engineered nanomaterials by the
environment may increase their toxicity. However, the
conclusions regarding cellular toxicity should not be confused
with the conclusions on the risk due to these NPs. If the
toxicity observed in Caco-2 cells with degraded
nanoparticles is slightly higher than that observed with the initial,
coated nanoparticles, the risk for humans (dose x
exposure) is clearly not increased, as the highest concentrations
to which humans may be exposed are currently 100,000
times lower than the concentrations tested .
Nevertheless, special care should be taken in designing new
nanoparticles, so that they do not transform into more
toxic compounds. It can be assumed that a safer design
of nanoparticles might include robust protective layers
Two protocols for NP alteration of the Nanobyk (BYK
Company) were used in this study. These conditions
reproduce extreme and long-term environmental conditions
of aging (100% hygrometry and permanent sunshine).
Before and after this process, a sample of the suspension was
centrifuged (200,000 g) and freeze-dried for fine structural
analysis . A Nanobyk stock suspension was diluted in
milliQ water at 220 mg/mL. Firstly, 500 mL of this
suspension was irradiated under artificial daylight for 112 days
using OSRAM HQI-BT lamps (E40, 400 W) with a
uniform spectral intensity between 425 and 650 nm, and
under continuous stirring (100 rpm). A 4 mg/mL stable
stock suspension of light-degraded Nanobyk (NB-DL)
was obtained after 4 months, the time necessary to reach
a stable conductivity and pH. This stock suspension of
NB-DL was then diluted in a culture medium to obtain
the exposure concentrations. Secondly, gastric
degradation of the Nanobyk 3810 was mimicked using a
simulated gastric medium (0.2% NaCl, HCl, pH = 1) for 3 h at
37C . This solution was then neutralized using
NaHCO3. The stock suspension obtained (NB-DA, 2 mg/
mL) was then diluted in the culture medium to obtain the
All concentrated suspensions were prepared in culture
medium and allowed to be in contact with the medium
prior to being diluted to exposure concentrations.
Physicochemical characterization of CeO2-based
The size, shape and mineralogy of Nanobyk NPs before
and after alteration, as well as of pristine CeO2 NPs
(originating from Rhodia), were characterized by
highresolution TEM, using a JEOL 2010 F operating at
200 kV. Samples were prepared by evaporating a droplet
of the suspension on a carbon-coated copper grid at
room temperature. The aggregation states of the
nanoparticles were measured by DLS (n = 3) in pure water
and in the culture medium, with and without 10% FCS,
using the Zetasizer nano ZS (Malvern Instruments Ltd,
UK). The zeta potential was also measured on the same
instrument. The crystal structure and Ce oxidation state
of CeO2 NPs and Nanobyk NPs were monitored on the
atomic scale by X-ray absorption spectroscopy at the
Ce L3-edge (5723 eV). Experiments were carried out in
transmission mode on the ELETTRA synchrotron
(Trieste, Italy) on the XAFS 11.1 beam line . Before and
after aging, the suspension was centrifuged (200,000 g)
and freeze-dried. The powders were then diluted in PVP
and pressed into thin pellets. The spectra were compiled
from the merging of three scans, and the energy was
calibrated using a standard reference CeO2. XANES (X-ray
absorption near-edge structure) data were obtained after
performing standard procedures for pre-edge
subtraction, and normalization using the IFEFFIT software
Cell culture and exposure conditions
Caco-2 cells (ATCC, Manassas, VA, US) were cultured
in Eagles Minimum Essential Medium supplemented
with 10% FCS (LGC Standards, Middlesex, UK) and
penicillin/streptomycin (100 g/mL) in a humidified
incubator at 37C, and 5% CO2. The cells were used
between passages 20 to 40. The cells were passaged weekly
at a seeding concentration of 6x103 cells/cm2 and the
medium was changed three times per week.
Measurement of transepithelial resistance (TEER)
For TEER measurements, Caco-2 cells were seeded at a
density of 5104 cells per well in 12 Transwell culture
plate inserts. MEM supplemented with 10% FCS was
added to the apical and basolateral chambers and
replenished three times a week. Cultures were confluent
at 4 days and stabilized maximum resistance values were
reached after 21 days. Transepithelial specific resistance
was measured at 37C using an STX2 electrode with an
EvomX recorder (WPI). Blanks (inserts without cells but
containing medium) were used to determine baseline
values of electrical resistance. Results were expressed in
ohms.cm2. Each experiment was repeated three times
and three measurements were made for each culture
Caco-2 cells were grown in 96-well plates and
differentiated for 21 days. The cells were exposed for 24 h or 72 h
to serially diluted concentrations of CeO2 NPs (21.25,
42.5, 85, and 170 g/mL, 100 L per well). Cells were
washed with PBS and cell viability was determined by
the ATP test as specified by the supplier (CellTiter-Glo
luminescent cell viability Assay, Promega). Briefly,
100 L kit reagent were added per well and the plate
was shaken for 10 min at RT before measuring
bioluminescence (LUMIstar Galaxy, BMG). Hydrogen
peroxide (2.5 mM, 1.25 mM, and 0.625 mM) was used as a
Caco-2 cells were grown in 96-well plates and
differentiated for 21 days. The cells were exposed for 24 h or 72 h
to serially diluted concentrations of CeO2 NPs (21.25 to
170 g/mL, 100 L per well). Cells were washed with
PBS and cell viability was determined by the XTT test as
specified by the supplier (In Vitro toxicology assay kit
XTT based, Sigma-Aldrich). Briefly, 20 L kit reagent
were added per well and the plate was incubated for 2 h
at 37C before reading absorbance at 450 nm and
690 nm (Multiscan Spectrum, Thermo Electron
Corporation). Hydrogen peroxide (2.5 mM, 1.25 mM and
0.625 mM) was used as a positive control.
Scanning Electron Microscopy (SEM)
Caco-2 cells were seeded at 5104 cells/cm2 in culture
medium on clear Millicell-24 Cell Culture Insert Plates
with a polyethylene terephthalate (PET) membrane
(Millipore) for SEM observation, and allowed to
differentiate for 21 days in a 5% CO2 incubator. The cells
were exposed to highly concentrated NB-type CeO2 NPs
(170 g/mL). After 72 h, the cells were washed three
times with PBS, fixed with 5% glutaraldehyde in 0.1 M
cacodylate for 1 h at 4C, then washed again twice with
distilled water and dehydrated in graded ethanol baths
(35, 70, 85, 95 and 100%). Finally, the cells were
dehydrated in HMDS (SPI-Chem) before examination by
SEM. The samples were metallized by deposition of
carbon (evaporation of braided carbon fiber (Agar scientific
Stansted, UK) then analyzed on Balzers MED010
(Balzers Union, Lichtenstein).
Microarrays and gene expression analysis
For microarray experiments, the cells were seeded in
6well plates (to collect sufficient amounts of RNA) in the
above medium at 5 104 cells/cm2 and allowed to
differentiate for 21 days. The cells were exposed to
21.25 g/mL for 72 h of all types of CeO2 NPs (3 mL/
well), in duplicate. The cells were also exposed to
hydrogen peroxide for 24 h at 20 M, as a positive control.
Control duplicates were achieved in the vehicle. Each
condition of exposure to NPs was compared with a
control, i.e. untreated cells (control 1 for NB and NB-DL,
control 2 for NB-DA, control 3 for hydrogen peroxide
and pristine CeO2). The cells were washed extensively
to avoid co-extraction of nucleic acids with NPs
adsorbed onto the cell surface, collected with trypsin
and washed with PBS. The cells were centrifuged and
RNA extracted using the RNeasy kit (Qiagen). The
RNAs were quantified with the Nanodrop 1000 and
their qualities analyzed on an Agilent Bioanalyzer
2100. The RNAs were amplified and labeled with
cyanine-3 fluorophore using a QuickAmp kit (Agilent),
according to the suppliers protocol. The efficiency of
fluorescent labelling was controlled by UV
spectroscopy (Nanodrop 1000) before hybridization on
commercial Agilent oligo microarrays (Human V1 4X
44 K) in technical duplicates.
The 44,000 spots represent probes of the whole
human genome, including redundancy. The microarrays
were scanned with a GenePix 4000B (Axon Instrument
Inc., Forster City, CA) in one-color mode at 532 nm and
5 m resolution. Each condition of exposure to NPs, as
well as controls, led to four hybridizations (two
biological replicates and two technical replicates). We used
Agilent microarrays with four independent genomes per
chip (4 44,000 probes), thus 8 microarrays are
sufficient to perform 32 hybridizations.
In this experimental design, six analyses were
conducted: a) control 2 versus control 1, as the experimental
negative control, b) hydrogen-peroxide-exposed cells
versus unexposed cells (named control 3), as the
experimental positive control, and c) surface-untreated
(pristine) CeO2-NP-exposed cells versus unexposed cells
(named control 3), d) NB-exposed cells versus
unexposed cells (named control 1), e) NB-DL exposed versus
unexposed cells (named control 1) f ) NB-DA exposed
versus unexposed cells (named control 2). For each
analysis, eight raw fluorescence data files were obtained
after scanning, which corresponded to four treated cells
and four control cells. All files (n = 32) were submitted
to GeneSpring software GX11 (Agilent Technologies)
for statistical analysis.
Concerning the statistics methodology, we used a
widespread method for determining the significance of the
change in gene expression [28,35]. The raw data were first
normalized using the percentile shift 75 normalization
method. The normalized data were then filtered on the
basis of spots present on 100% of the slides in one of two
conditions (treated NPs or control). Only spots detected
with at least 70% of their pixels above the threshold
intensity signal (set to the median background plus two
standard deviations) were selected. From the remaining spots,
we selected those with fluorescence ratios (representing
NP-exposed samples versus unexposed samples) greater
than a 1.5-fold-change cut-off, then we determined the
statistical significance of the changes with a p-value 0.05
using a Students t-test statistical analysis on Genespring
software and performing a Benjamini and Hochberg false
discovery rate multiple testing correction. We thus
obtained probe sets which were significantly induced or
repressed after exposure to various NPs. See Additional
file 4: Table S1 in the Supporting Information paragraph.
Data were analyzed through the use of IPA (Ingenuity
Systems, www.ingenuity.com). Canonical pathways
analysis identified the pathways from the IPA library of
canonical pathways that were most significant to the data
set. Molecules from the dataset that met the fold change
cut-off of 1.5 with p-value <0.05, and were associated
with a canonical pathway in the Ingenuity Knowledge
Base, were considered for the analysis. The significance
of the association between the dataset and the canonical
pathway was measured in two ways: 1) a ratio of the
number of molecules from the dataset that map to the
pathway divided by the total number of molecules that
map to the canonical pathway is displayed; 2)
righttailed Fishers exact test was used to calculate a p-value
determining the probability that the association between
genes in the dataset and the canonical pathway is
explained by chance alone.
Total RNA was isolated according to the
manufacturers instructions using the RNeasy kit (Qiagen) and
treated with DNase. RNA purity and concentration
were determined by UV on a Nanodrop
Spectrophotometer and integrity was assessed on an Agilent 2100
Bioanalyzer (Agilent Technologies). All the samples
used in this study showed a 28S/18S ratio indicating
intact and pure RNA. Differential analysis of RNA
from cells exposed to NPs and unexposed cells was
performed by qRT-PCR with the Sybr Green PCR
Master Mix (Finzyme) kit, according to the manufacturers
instructions on Opticon II (Biorad). Primer (Sigma)
sequences were, for ATP5J: 5 GTCAGCCGTCTCAG
TCCATT 3 (forward) and 5 AAAAGCTCCCTCTCC
AGCTC 3 (reverse); for NDUFA4: 5 TCCAGATGT
TTGTTGGGACA 3 (forward) and 5 GTGGAAAA
TTGTGCGGATGT 3 (reverse); for NDUFS7: 5 CG
CAAGGTCTACGACCAGAT 3(forward) and 5 TCC
CGCTTGATCTTCCTCT 3 (reverse); for PRDX5: 5 G
TGGTGGCCTGTCTGAGTGT 3 (forward) and 5 ATG
CCATCCTGTACCACCAT 3 (reverse); for PRDX3: 5 G
TTGTCGCAGTCTCAGTGGA 3 (forward) and 5 GA
CGCTCAAATGCTTGATGA 3 (reverse); for COX6A2:
5 CTACCAACACCTCCGCATC 3 (forward) and 5 TC
GAAGCTTCACACCTTTATTG 3 (reverse); for SDHC:
5TTGAGTGCAGGGGTCTCTCT 3 (forward) and 5 A
ACCAGGACAACCACTCCAG 3 (reverse); for SDHD:
5 GTATGCCTCTTTGCCTCTGC 3 (forward) and 5
GAGGCAACCCCATTAACTCA 3 (reverse). For ATP5J,
NDUFA4, NDUFS7, PRDX5, PRDX3, COX6A2 , SDHC
and SDHD, the amplicon sizes were 186, 199, 241, 203,
216, 155, 240, and 203 bp, respectively. The
measurements were the means of three independent experiments,
and normalization was based on the total RNA mass
quantified on the Nanodrop.
Availability of supporting data section
The data sets supporting the results of this article are
available as additional files (Additional file 4: Table S1).
The raw data discussed in this publication have been
deposited in NCBIs Gene Expression Omnibus (GEO)
repository and are accessible through GEO Series accession
number GSE60128 (http://www.ncbi.nlm.nih.gov/geo/
Additional file 1: Figure S1. Experimental XANES spectra at the Ce
L3-edge of CeO2 NPs, the unaltered Nanobyk, light-degraded Nanobyk
(Nanobyk DL) and acid-degraded Nanobyk (Nanobyk DA), nano-sized CeO2,
and micron-sized CeO2. No change in the cerium redox state was observed
between initial and altered Nanobyk.
Additional file 2: Figure S2. SEM image of Caco-2 cells exposed for
72 h to 170 g/mL NPs. Caco-2 cells were grown on bicameral wells
(PET, pores 1 m) and differentiated for 21 days. The cells were exposed
to NPs (170 g/mL). After 72 h incubation, the cells were washed, fixed
and dehydrated. They were observed by SEM. Top lane) Magnification
2,000 x. Bottom lane) Magnification 16,000 x. Clear spot deposits are visible
at the cell surface only for light-degraded Nanobyk.
Additional file 3: Figure S3. Microarray scatter plots. Caco-2 cells were
cultured and differentiated for 21 days. They were exposed for 72 h to
21.25 g/mL CeO2 NPs. The scatter plots represent the raw fluorescence
intensities of genes filtered at the threshold intensity signal after
hybridization (n = 4). From Blue to Red: increasing fluorescence intensity.
The number of genes detected above the signal threshold was compared
for each type of NP (y-axis) versus their own control (x-axis). A) Unexposed
cells versus unexposed cells (control 2 versus control 1) as negative control.
B) H2O2-exposed cells versus unexposed cells (control 3) as positive control.
C) Pristine (surface-untreated) cerium-oxide-NP-exposed cells versus
unexposed cells (control 3). D) NB-exposed cells versus unexposed
cells (control 1). E) NB-DL-exposed cells versus unexposed cells (control 1).
F) NB-DA-exposed cells versus unexposed cells (control 2). These graphs do
not display the significantly altered genes since they represent the raw
fluorescence signals before applying statistical tests. Nevertheless, they
give a good, rough overview of the amplitude alterations caused by
the different nanoparticles.
Additional file 4: Table S1. Complete lists of significant, differentially
expressed genes including fold change and p-value in each probe set.
NA means no available information.
Additional file 5: Table S2. List of 37 genes common to NB-DL and
NPs: Nanoparticles; NB: Nanobyk; NB-DL: Nanobyk nanoparticles degraded by
light exposure; NB-DA: Nanobyk nanoparticles degraded by acidic treatment
with SGF; SGF: Simulated gastric fluid; RT: Room temperature; TEER: Trans-epithelial
electric resistance; SEM: Scanning electron microscopy; TEM: Transmission electron
microscopy; FCS: Fetal calf serum; PBS: Phosphate-buffered saline; ROS: Reactive
oxygen species; HMDS: Hexamethyldisilazane; d: Inter-reticular distance;
Dh: Hydrodynamic diameter; PVP: Polyvinylpyrrolidone; EDX: Energy-dispersive X-ray
spectroscopy; BSE: Back-scattered electron detector; qRT-PCR: Real-time quantitative
polymerase chain reaction.
MF carried out the cell culture, viability studies, size determination, and
cellular SEM studies, participated in the microarray experiments and drafted
the manuscript. GS performed RNA extraction, labelling and microarray
experiments. FB carried out the statistical analyses and bioinformatics studies.
MA performed the physicochemical characterization of NPs including TEM
and XANES. JR coordinated the different teams taking part in the whole
project and provided the NPs. OP conceived the study, participated in
design and experiments, coordinated the team and wrote the final
manuscript. All authors read and approved the final manuscript.
The authors wish to thank the Agence Nationale de la Recherche for
funding the AgingNano&Troph project (ANR-08-CESA-001). Technical help for
SEM studies was provided by C. Dominici, CP2M platform, Universit
Aix-Marseille. We also thank J. Courageot, Universit Aix-Marseille for
1CEA, IBEB, SBTN, Laboratoire dEtude des Protines Cibles, F-30207
Bagnols-sur-Cze, France. 2CEREGE, UMR 7330 CNRS/Aix-Marseille Universit,
ECCOREV, Europle de lArbois, F-13545 Aix-en-Provence, France.
3International Consortium for the Environmental Implications of
Nanotechnology (iCEINT), Aix-en-Provence, France.
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