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Genotoxic Impact of Aluminum-containing
Nanomaterials in Human Intestinal and Hepatic
Cells.
Pégah Jalili
Anses Laboratoire de Fougeres
Sylvie HUET
Anses Laboratoire de Fougeres
Agnès Burel
MRiC
Benjamin-Christoph Krause
Bundesinstitut fur Risikobewertung
Caroline Fontana
Institut National de Recherche et de Securite
Soizic Chevance
Institut des Sciences Chimiques de Rennes
Fabienne Gauffre
Institut des Sciences Chimiques de Rennes
Yves Guichard
Institut National de Recherche et de Securite
Alfonso Lampen
Bundesinstitut fur Risikobewertung
Peter Laux
Bundesinstitut fur Risikobewertung
Andreas Luch
Bundesinstitut fur Risikobewertung
Kevin Hogeveen
Anses Laboratoire de Fougeres
Valerie Fessard ( Valerie.FESSARD@anses.fr )
Anses Laboratoire de Fougeres https://orcid.org/0000-0001-9046-9117
Research
Keywords: nanomaterials, comet assay, micronucleus, cell transformation assay, oxidative stress
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Abstract
Background: Exposure of consumers to aluminum-containing nanomaterials (Al NMs) through numerous
products is an area of concern for public health agencies since human health risks are not completely
elucidated. In addition, the available data on the genotoxicity of Al2O3 and Al0 NMs are inconclusive or
rare. In order to provide further information, the present study investigated the
in vitro
genotoxic potential
of Al0 and Al2O3 NMs in intestinal and liver cell models since these tissues represent organs which would
be in direct contact or could experience potential accumulation following oral exposure.
Methods: Differentiated human intestinal Caco-2 and hepatic HepaRG cells were exposed to Al0 and
Al2O3 NMs (0.03 to 80 µg/cm2) and the results were compared with those obtained with the ionic form
AlCl3. Several methods, including H2AX labelling, the alkaline comet assay and micronucleus (MN)
assays were used. Oxidative stress and oxidative DNA damage were assessed using High Content
Analysis (HCA) and the formamidopyrimidine DNA-glycosylase -modied comet assay respectively.
Moreover, carcinogenic properties of Al NMs were investigated through the cell transforming assay (CTA)
in Bhas 42 cells.
Results: The three forms of Al did not induce chromosomal damage when tested in the MN assay.
Furthermore, no cell transformation was observed in Bhas 42 cells. However, although no production of
oxidative stress was detected in HCA assays, Al2O3 NMs induced oxidative DNA damage in Caco-2 cells
in the comet assay following a 24 h treatment. Considerable DNA damage was observed with Al0 NMs in
both cell lines in the comet assay, although this was likely due to interference with these NMs. Finally, no
genotoxic effects were observed with AlCl3.
Conclusion: The slight effects observed with Al NMs are therefore not likely to be related to ion release in
the cell media.
1. Introduction
Within the last decade, aluminum (Al)-containing nanomaterials (NMs) have been widely used not only
for industrial applications, but also in consumer products, due to their higher reactivity compared to the
bulk form [1, 2, 3, 4]. Forms of Al, both in the micro-and the nano-size, are present in food and consumer
products [1, 5] due to their use as rming, anticaking, neutralizing, emulsifying and texturizing agents, as
well as for cooking tools [6], waste water treatment [7, 8] and in medical and hygiene products such as
toothpaste [9, 10, 11]. Nevertheless, their potential toxicity has not been fully evaluated, leading to major
concerns from consumers and public health agencies [12].
According to exposure estimates from the European Food Safety Authority (EFSA), consumers can
absorb up to 2.3 mg Al /kg bw/week, more than twice the weekly tolerable intake (1 mg/kg bw/week) [7].
In addition, a recent study has estimated total consumer exposure to Al containing compounds, including
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contributions from products used in food (additives, contact materials) and in cosmetics, and concluded
that adolescents were highly exposed [13].
Few studies on the genotoxicity of nanoscale forms of Al following oral ingestion have been performed,
and most of the published literature has focused on Al2O3 NMs only. DNA damage was reported in
erythrocytes of rats after a single oral treatment with Al2O3 NMs, although at high doses (≥1,000 mg/kg)
[14, 15]. Genotoxic effects were observed in bone marrow, but not in other organs, after a short-term
treatement with lower doses of Al2O3 NMs [16].
In vivo
effects of Al0 NMs following oral exposure are
mostly lacking, although one study suggested cross-linking effects on DNA in the duodenum of rats [16].
Following oral exposure of rodents with ionic forms of Al, an increase in MN frequency was reported in
bone marrow after a single oral administration [17] and in liver after a 30 day oral treatment [18].
Nevertheless, the induction of MN formation in liver was shown to decrease with an antioxidant treatment
[18, 19]. Consistent with these results, a slight oxidative DNA damage was observed in blood after a short-
term oral exposure [16].
The
in vitro
genotoxicity of Al2O3 NMs has been assessed in several mammalian cell lines including
human peripheral lymphocytes [20], primary human broblasts [21], hepatic HepG2 cells [22], and
Chinese hamster ovary cells [23]. While some studies have not observed genotoxic effects of Al2O3 NMs
[20, 24, 25, 26], others have reported a positive response [21, 22] which may be associated with oxidative
damage [22]. In contrast, no data on the
in vitro
genotoxicity of Al0 NMs has been published so far, and
only some cytotoxicity was detected in rat alveolar macrophages treated with Al0 above 100 µg/ml [27].
For the salt AlCl3, DNA damage has been reported in human peripheral blood lymphocytes, with positive
results in micronucleus and chromosomal aberration tests, as well as in the comet assay [17, 28, 29, 30].
According to an ECHA safety assessment [31], the data available on the genotoxicity of Al2O3 NMs are
inconclusive while few data on the genotoxicity of Al0 NMs has been published so far. In addition to the
direct contact of Al NMs present in food with the intestinal epithelium, Al accumulation in liver has been
shown after oral exposure with Al2O3 NMs [15, 32, 33].
Therefore, the aim of the current study was to evaluate the
in vitro
genotoxic potential of Al0 and Al2O3
NMs in two relevant human cell models of intestine and liver. Several endpoints of genotoxicity were
investigated using the alkaline and Fpg-modied comet assays which detects DNA breakage including
oxidative lesions, DNA double strand breaks were detected through phosphorylated histone H2AX
(γH2AX), and the micronucleus assay which determines chromosome and genome damage.
Furthermore, the capacity of aluminum-containing NMs to initiate or promote carcinogenesis was
assessed by the Cell Transforming Assay (CTA) in Bhas-42 cells.
As these NMs can potentially dissolve in the dispersion solution or in media, the genotoxicity was
compared to that of the metal salt AlCl3. Moreover, the interference of NMs, including with Al-NMs [34],
has been demonstrated in numerous publications using a wide range of biological assays, and stresses
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the necessity to evaluate interference in order to assess the potential effect on the results [35, 36, 37, 38].
In this study, various sources of interference have been taken into account within the different assays.
2. Materials And Methods
2.1 Chemicals and reagents
Dimethylsulfoxide (DMSO), insulin, cytochalasin B, formamidopyrimidine-DNA glycosylase (Fpg), trypan-
blue, 12-
O
-tetradecanoylphorbol-13-acetate (TPA), 3-methylcholanthrene (3-MCA) and menadione (MEN)
were supplied from Sigma (St. Quentin-Fallavier, France). Methylmethanesulfonate (MMS) was
purchased by Acros Organics (Fairlawn, NJ). Dinophysistoxin-2 (DTX-2) was from the National Research
Council Canada (NRCC, Ottawa, Canada). Penicillin, streptomycin, Williams' E medium and Fetal Bovine
Serum Fetalclone II (FBS) were supplied from Invitrogen Corporation (Illkirch, France). For Bhas 42 cell
cultures, Eagle’s minimum essential medium and Dulbecco’s modied Eagle’s medium/Ham’s F12 was
from Invitrogen Corporation (Illkirch, France). Fetal bovine serum was obtained by Dutscher, (Brumath
France). Hydrocortisone hemisuccinate, HycloneTM DMEM/high glucose and fetal bovine serum for Caco-
2 cells were purchased from Upjohn Pharmacia (Guyancourt, France), GE Healthcare Life Science (Logan,
UT, USA) and Capricorn scientic (Ebsdorfergrund, Germany), respectively. The primary and secondary
antibodies (mouse monoclonal anti γH2AX ser139 (ab26350), rabbit monoclonal anti active caspase-3
antibody (ab13847), goat anti-rabbit IgG H&L AlexaFluor 647 (ab150079) and goat anti-mouse IgG H&L
AlexaFluor 647 (ab150115)) were provided from Abcam (Cambridge, UK). CellROX® Deep Red Reagent
was obtained from Invitrogen (Paisley, UK). Formaldehyde and Giemsa were purchased by Fisher (Illkirch-
Graffenstaden, France).
2.2 Dispersion and characterization of NMs
Al0, Al2O3 and ZnO NMs with a similar primary particle size were supplied from IoLiTec (Heilbronn,
Germany). NM characteristics as provided by the supplier are presented in Table 1. AlCl3 (hexahydrate)
was purchased from Sigma Aldrich (Saint Louis, USA). NM dispersion was performed according to the
NANOGENOTOX protocol [39], as described in [16].
The morphology and agglomeration of Al0 and Al2O3 NMs in the stock dispersion solution and in cell
media were determined by transmission electron microscopy (TEM) (Figure S 1). For the characterization
of NMs from stock solutions, TEM grids were prepared immediately after sonication and dilution (100
µg/mL) in the stock dispersion solution. For the characterization of NMs in cell culture media (DMEM
+10% FBS and William’s Medium +5% FBS), the samples were diluted with distilled water to 1.2 µg/mL
prior to grid preparation. The TEM grids were prepared by deposition of a carbon-coated copper grid onto
a drop of the stock solution for 20 s to allow adsorption of the NMs and were observed with an electron
microscope (JEOL 1400 operated at 120 kV and coupled with a 2k-2k camera from Gatan (Orius 1000)).
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The hydrodynamic diameter of Al0 and Al2O3 NMs were measured using a Malvern Zetasizer (Malvern
Instruments, Malvern, UK) equipped with a 633-nm laser diode operating at an angle of 173°. To assess
the stability of NM suspensions, following NM dispersion, samples were diluted to a nal concentration
of 100 µg/ml in the stock dispersion solution or in cell media and measurements were performed at 0
and 24 h. The samples were equilibrated at 25 °C for 120 s prior to measurement. Ten repeated
measurements for each sample were performed in 3 independent experiments. The mean hydrodynamic
diameter Zave was determined using cumulant analysis.
2.3 Cell culture and treatment
The human colorectal adenocarcinoma Caco-2 cell line was cultured (passages 25–38) until
differentiation after 21 days as described in [40] including for cell seeding in various plate formats
depending on the assay performed. Simarly, HepaRG cells (passages 13-19) were cultured and seeded for
the various assays as previously described [40, 41].
Differentiated Caco-2 and HepaRG cells were treated for 24 h with Al0 and Al2O3 NMs at concentrations
ranging from 0.03 to 80 µg/cm2 and with AlCl3 as ionic salt control at 90 and 128 µg.mL-1 in DMEM +
10% FBS or William’s medium + 5% FBS respectively. For some assays, ZnO NMs at concentrations from
1.5 to 6 µg/cm2 were used as a positive NM control. Equivalence between volume concentration (µg/mL)
and surface concentration (µg/cm2) are shown in Table S 1B. Al content corresponding to the
concentrations of Al-containing NMs and AlCl3 that were used are summarized in Table S 1B.
2.4 Kinetics of nanoparticle sedimentation
The colloidal characterization of the suspended nanomaterials in the conditions of cellular uptake assay
was achieved using the volumetric sedimentation method (VCM) as reported in DeLoid et al [42]. We rst
measured the volume of the potentially agglomerated NM in DMEM and Williams media, at a NM
concentration of 250 µg.mL-1, using a specic centrifugal tube and ruler device. From the measured pellet,
the effective density (eff) is calculated using the following equation:
Where m is the density of the medium in g.cm-3, NP is the density of NP (2.7 g.cm-3 for Al and 3.95 for
Al2O3), MNP the total mass of NM in 1 mL of dispended volume and V the measured volume pellet. SF is
a stacking factor and was set to 0.634, which generally is appropriate for random stacking. The loss of
mass of NMs from ion release was estimated to be lower than 1% and was neglected in the density
calculation. The viscosity of the cell culture media at 37°C was determined using a Nanoparticle Tracking
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Analysis device (Malvern Instrument) by measuring the apparent hydrodynamic radius of 400nm
standard particles in the media. Finally, the kinetics of sedimentation was calculated using the distorded
grid (DG) model available from DeLoid et al [42]. The size of the NPS was taken from Table 2 (Zave).
Other model parameters are h=3.1mm (liquid column height), initial NM concentration : 0.250mg.mL-1,
the dissolution and cell-NMs stickiness are neglected (parameters set to 0).
2.5 Ion release from NMs
Following the dispersion of Al0 and Al2O3 NMs, suspensions were diluted in stock solution (ultra pure
water + 0.05 % BSA) or cell culture media (DMEM +10% FBS and William’s Medium +5% FBS) at
concentrations of 25, 50 and 100 µg/mL. After 24 h, ion release from NMs was determined by
ultracentrifugation at 16,000g for 1h at 4°C (Hettich Zentrifuge Mikro 220R). The supernatants were
processed through acidic hydrolysis (69% HNO3, 180°C for 20 min in an MLS-ETHOS Microwave system)
before detection of Al species with a quadrupole Inductively Coupled Plasma Mass Spectrometry (ICP-
MS) (iCAP Q, Thermo Fisher Scientic GmbH, Dreieich, Germany) equipped with a PFA ST Nebulizer, a
quartz cyclonic spray chamber and a 2.5 mm quartz injector (Thermo Fisher Scientic). The gas ows
were set to 14 L/min, and 0.65 L/min for the cool gas (Ar) and the auxiliary gas (Ar) respectively. The ow
rate of the sample was 0.39 mL/min. Results are given as percentage of the initial Al amount.
2.6 Uptake observations by TEM
Following a 24 h treatment, cells were xed by glutaraldehyde (2.5%) and embedded in DMP30-epon
before cutting ultra-thin sections (90 nm) for TEM observation as described in [40].
2.7 Cellular imaging and High Content Analysis (HCA)
After 24 h treatment with Al NMs and AlCl3, plates were processed for HCA with an ArrayScan VTI
HCS Reader (Thermo Scientic, Waltham, USA) as described in [40]. Cell numbers were determined from
DAPI staining, active caspase-3 was quantied in the total cell compartment and H2AX in cell nuclei.
Oxidative stress was measured using CellROX Deep Red Reagent (Fisher Scientic, Illkirch, France).
Briey, cells were pre-incubated for 1 h with 5 M CellROX in serum-free media and washed twice with
PBS before treatment with NMs and AlCl3. After 24 h and twice washing with PBS, cells were incubated
with 3 M Hoescht 33342 for 20 min at 37°C. Cells were then washed twice with PBS and were scanned
and analyzed using the Compartimental Analysis module of the Bioapplication software. For each well,
images from 7 elds (20 × magnication) were analyzed for quantication of uorescence at 647 nm.
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2.8 Comet assay
After a 5 h (Figure S 3) or 24 h treatment with Al NMs and AlCl3, the comet assay was performed as
described in [40, 43]. The individual tail intensity of at least 50 cells per slide were analyzed using the
Comet Assay IV software (Perceptive Instruments, Haverhill, UK). Cells were considered as hedgehogs
when DNA damage was too high to score. At least three independent experiments were performed. Methyl
methanesulfonate (MMS) was used as positive control.
The level of oxidized bases was determined with the modied comet assay using the bacterial DNA repair
enzyme Fpg through the formation of single-strand breaks (SSB) induced by the excision of oxidized
purines [44, 45]. Some additional steps to the protocol described above were performed such as
incubation with enzyme buffer (0.1 M KCl, 0.2 mM EDTA, 40 mM HEPES, 0.2 mg/ml BSA) after lysis.
Two slides, one incubated with enzyme buffer (control slide) and the other with 9 U/slide Fpg at 37°C for
30 min, were then processed as described previously.
2.9 Particle interaction with DNA during the comet assay
The interaction of NMs with DNA migration during the comet assay was evaluated as described
previously [35, 40]. Briey, dilutions of Al0 or Al2O3 NMs in 0.5% low-melting point agarose (LMP) were
prepared at nal concentrations of 28 and 128 µg/mL (corresponding to 9 and 40 µg/cm2 conditions).
After trypsinization and centrifugation (2 min, 136 g), untreated Caco-2 and HepaRG cells were
resuspended in the LMP/NM mixture, loaded on pre-coated slides and processed in the alkaline comet
assay as previously described, in the presence or absence of Fpg. A negative control consisting of
untreated cells in LMP-agarose in the absence of NMs was performed in order to compare the results.
2.10 Cytokinesis-block micronucleus assay (CBMN)
The CBMN assay was performed as described in [40] according to the guideline n°487 of the
Organization for Economic Co-operation and Development (OECD) [46]. After staining of the slides with
acridine orange (100 μg/mL), at least 1000 binucleated cells per slide were scored. Three independent
experiments were carried outand each concentration was tested in duplicate. The replication index (RI)
was calculated using the formula recommended by OCDE guideline n°487. MMS and ZnO NM were used
as positive controls.
2.11 Bhas 42 Cell Transformation Assay (CTA)
Originally established from the v-Ha-ras-transfected BALB/c 3T3 cells by Sasaki et al [47], Bhas 42 cells
used in this study (passage 23) were obtained from Harlan Laboratories (Rossdorf, Germany). Both the
CTA and concurrent cell growth assays were performed in their 6-well format and in accordance with a
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guidance document produced by the OECD [48], with some modications. The protocol, including both an
initiation and a promotion assay, was previously described by Fontana et al [49].
In the initiation assay, 24 h after seeding (420 cells/cm2) (Day 1), the cells were treated with Al NMs and
AlCl3 for 72 h (Day 4). Then, the cells were cultivated in fresh medium until Day 21, with medium changes
on Day 7, Day 10 and Day 14. MCA (1 µg/mL) was used as positive control.
In the promotion assay, the cells were seeded (1,500 cells/cm2) and cultured for 4 days without changing
the media. On Day 4, 7, and 10, the culture medium was replaced with fresh media containing Al NMs or
AlCl3. The treatment continued until Day 14. The cells were then cultured in fresh medium in the absence
of NMs until Day 21. TPA (0.05 µg/mL) was used as positive control.
In both assays, the cells were xed with ethanol on Day 21 and stained with a 5 % Giemsa solution. The
morphological criteria recommended by OECD were followed for the evaluation of transformed foci. The
mean of the number of transformed foci was calculated from six replicate wells.
Cell growth assays in both the initiation and promotion conditions were performed on Day 7 using three
replicate wells for each condition. The cells were xed in 4% formaldehyde and stained with 1 µg/mL
DAPI. The number of cell in wells was determined by automated microscopy with an Arrayscan VTi using
the Target Activation module of the BioApplication software. The relative cell growth (%) was calculated
as follows: (number of cells in treated cultures / number of cell in control cultures) x100.
2.12 Statistical analysis
The statistical signicance of HCA results was tested using one-way Analysis of variance (ANOVA)
followed by Dunnett's post-hoc tests with GraphPad Prism 5.
For the comet assay, the one-way Analysis of variance (ANOVA) was used followed by Dunnett's post-hoc
test.
For the micronucleus assay, the percentages of micronucleated cells in treated and solvent control
cultures were compared using the one-way Pearson chi-square test.
For the CTA, data were statistically analysed by multiple comparison using the one-sided Dunnett's test
(p<0.05, upper-sided). The signicance of the positive controls (MCA and TPA) was evaluated relative to
the control (p < 0.05) by the one-sided Student's t-test.
3. Results
3.1 Nanomaterial characterization
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Information concerning the physico-chemical characterization, including the morphology, primary size,
surface specic area (SSA), purity and density of the Al0, Al2O3 and ZnO NMs used in this study are
provided in Table 1. However, in contrast to the information provided by the suppliers, the particle
morphology of Al2O3 NMs in the stock dispersion solution cannot be considered as being spherical, but
rather have a rod-like shape when observed by TEM (Figure S1). Although Al0 particles exhibit a spherical
shape, numerous elongated protrusions are also observed (Figure S1). Therefore, values of “average
particle size” (Table 1) should then be considered with caution. Due to the drying step for preparation of
TEM grids, the crystallization of different components of the culture media did not allow a proper
characterization of the morphology of Al NMs in cell culture media (data not shown).
Particle hydrodynamic diameter and stability in the stock dispersion solution, as well as in cell media,
were assessed by DLS immediately (0 h), as well as after 24 h (Table 2). The hydrodynamic diameters of
Al0, Al2O3 and ZnO NMs in the dispersion stock solution were 254 ± 4 nm for Al0, 168 ± 3 nm for Al2O3
and 233 ± 11 nm for ZnO immediately following dispersion and were stable over time. The stability over
time of these NMs was also observed in cell media (Table 2). Nevertheless, the average hydrodynamic
size of Al0 and Al2O3 NMs were lower in DMEM + 10% FBS at any time of measurement compared to
stock solution and William’s media + 5% FBS medium. The average hydrodynamic size of ZnO NMs was
similar in the stock dispersion solution and in the two media. Globally, although estimated to have a
similar primary size, the average hydrodynamic sizes of Al2O3 NMs are consistently smaller than those
measured for Al0 and ZnO NMs.
The polydispersity index (PdI) of Al0, Al2O3 and ZnO NMs suspensions in stock dispersion solution and in
media were stable over time. Whereas the PdI was quite low (< 0.25) for all NMs in stock dispersion
solution, it increased in cell media for Al2O3 (0.52 ± 0.027 in DMEM and 0.47 ± 0.015 in William’s at 0 h)
as well as for ZnO (0.27 ± 0.010 in DMEM and 0.23 ± 0.030 in William’s at 0 h). This effect of media on
the PdI was not observed for Al0 NMs.
The sedimentation of particles during
in vitro
exposure is critical when considering interactions of cells
with NMs. Indeed, in a typical experiment the NMs are dispensed onto adherent cells in well plates.
Therefore the amount of particles in contact with cells depends on the rate of sedimentation. We applied
the dosimetry method reported by DeLoid et al [42] to evaluate the sedimentation of Al0 and Al2O3 NMs in
DMEM and Willam’s media in the conditions of cell exposure. When dispersed in culture medium, NMs
may form agglomerates with adsorbed proteins and entrapped uid. The effective density (eff) of these
agglomerates should rst be measured to determine the colloidal behavior of such agglomerates. The
effective densities determined for Al0 NMs are 1.18 and 1.19 in DMEM and William’s media respectively.
Densities of 1.24 and 1.17 were obtained for Al2O3 NMs in DMEM and William’s cell culture media
respectively. Applying the sedimentation model provided in DeLoid et al [42], we calculated the evolution
of the concentration of NMs at the surface of cells with respect to time (Figure 1). The sedimentation of
Al0 NMs was similar in both media, and after 24 h the concentration at the bottom of the well is
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approximately 650 µg/mL. The rate of sedimentation of Al2O3 NMs was relatively slower, and the
difference was signicantly more pronounced in William’s medium, with a deposited concentration of 450
µg/mL at the bottom of the well after 24 h.
3.2 Ion release in stock solution and cell culture media
Ion release from Al NMs was investigated using ICP-MS (Table 3) and results are presented as
percentages with respect to the initial concentration of aluminum. A decrease in the percentage of ion
release from Al0 NMs was observed with increasing NMs concentrations in both the stock dispersion
solution (1.30 % at 25 µg/mL and 0.48 % at 100 µg/mL) and in media (3.88 % to 0.95 % in DMEM, and
2.42 % to 0.68 % in William’s for 25 and 100 µg/mL respectively). Nevertheless, ion release from Al0 NMs
was slightly higher in media when compared to the dispersion stock solution. A concentration-dependent
decrease in ion release was also observed for ZnO NMs (Table 3). Ion release was also higher in media
compared to dispersion stock solution.
In contrast to Al0 and ZnO NMs, for Al2O3 NMs, the percentage of ion release with respect to the initial
concentration was very low, relatively stable and independent of the NM concentration, although ion
release was slightly higher in cell media.
The level of ions from AlCl3 solutions was stable and independent of the concentration in the stock
solution, but decreased with increasing concentration in media. This decrease of ion concentration with
higher concentrations is likely due to the precipitate formed by AlCl3 in cell media.
3.3 Uptake of Al NMs in Caco-2 and HepaRG cells
The uptake and the intracellular distribution of Al0 and Al2O3 NMs following a 24 h treatment in Caco-2
and HepaRG cells were investigated by TEM (Figure 2 and 3). In both cell lines, the majority of Al0 NMs
were found as dense agglomerates of various sizes in the cytoplasm embedded in electron lucent or
dense vesicles which are likely endosomes and lysosomes (Figure 2 and 3, B and C, notched arrows).
Moreover, in certain cases, some Al0 NMs were observed as isolated nanoparticle clusters in the
cytoplasm proximal to the nucleus (Figure 2 and 3 C, full arrows). Observations of Al0 NMs in the nucleus
were very rare, and this result requires further investigation as it may be due to artefacts. The distribution
pattern observed for Al2O3 NMs was similar in both cell lines. While a perinuclear localization of Al2O3
NMs was also seen, this occured less frequently than for Al0 NMs (Figure 2 and 3, D and E). Even at the
lowest concentration, Al0 and Al2O3 NMs were internalized through vesicle formation, most likely
endocytosis, and accumulated in the cytoplasm of Caco-2 and HepaRG cells (Figure S 2).
3.4 Cytotoxicity
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Viability and apoptosis in Caco-2 and HepaRG cells following a 24 h treatment with Al NMs were
investigated by cell counts (Figure 4 A) and active caspase-3 labeling (Figure 4 B) respectively using
automated image analysis. No signicant change in cell numbers and active caspase-3 levels were
observed in either Caco-2 or HepaRG cells treated with Al0 and Al2O3 NMs up to 80 µg/cm2. In addition,
no signicant cytotoxic effects were observed in cells treated with the ionic salt control AlCl3 up to 128
µg/mL Al content (1.16 mg/mL AlCl3).
Similarly, no signicant change in cell numbers were observed in Caco-2 cells treated with ZnO at the
concentrations tested. However, a signicant decrease in cell numbers as well as an increase in active
caspase-3 levels was observed for the highest dose (6 µg/cm2) of ZnO NMs in HepaRG cells.
3.5 Oxidative stress
Quantication of intracellular ROS was used to evaluate oxidative stress in Caco-2 and HepaRG cells
following a 24 h treatment (Figure 5). Intracellular reactive oxygen species (ROS) levels were not
signicantly changed following treatment up to 80 µg/cm2 with Al0 and Al2O3 NMs or the ionic salt
control AlCl3. However, treatment with ZnO NMs signicantly increased levels of ROS at the highest
concentration (6 µg/cm2) in HepaRG cells.
3.6 Genotoxicity
3.6.1 γH2AX
Quantication of γH2AX labeling was used to evaluate the induction of DNA double stand breaks in
Caco-2 and HepaRG cells following a 24 h treatment with Al NMs. Compared to untreated cells, the
γH2AX levels were not affected in the nuclei of Caco-2 cells treated for 24 h with Al0 and Al2O3 NMs up to
80 µg/cm2, with ZnO NMs, or with the ionic salt control AlCl3 up to 128 µg/mL (Figure 6). However in
HepaRG cells, Al0 NMs induced a slight but statistically signicant increase in γH2AX levels at the highest
concentration (80 µg/cm2) tested. ZnO NMs induced signicant increases at 3 and 6 µg/cm2.
3.6.2 Comet assay
The potential for Al0 and Al2O3 NMs to induce DNA damage in Caco-2 and HepaRG cells was investigated
with the alkaline comet assay after a 24 h treatment (Figure 7 A and B). A modied comet assay with the
Fpg enzyme was also performed to detect oxidative DNA damage (Figure 7 C and D).
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In Caco-2 cells, a signicant increase in tail DNA was observed with Al0 NMs from 28 to 80 µg/cm2 in the
alkaline comet assay (Figure 7 A). In contrast, neither Al2O3 and ZnO NMs, or the ionic salt control AlCl3
induced any signicant increase in tail DNA. In the Fpg-modied comet assay, a signicant increase in
tail DNA was observed in cells treated with Al2O3 NMs at 3, 9 and 80 µg/cm2 (Figure 7 C).
In HepaRG cells, tail DNA signicantly increased in a dose-dependent manner in cells treated with Al0
NMs, including a very considerable effect starting at 28 µg/cm2. In contrast, no effect was observed for
cells treated with Al2O3 and ZnO NMs, or the ionic control AlCl3 (Figure 7 B). Similarly, an increase in tail
DNA in the Fpg-modied comet assay was observed for Al0 NMs at all concentrations tested with a very
signicant effect observed at concentrations above 9 µg/cm2. No signicant changes in tail DNA were
detected in HepaRG cells treated with Al2O3 and ZnO NMs or AlCl3 (Figure 7 D).
DNA damage in Caco-2 and HepaRG was also investigated by the alkaline comet assay after a 5 h
treatment (Figure S 3). In Caco-2 cells, a concentration-dependent increase in tail DNA was observed in
cells treated with Al0 NMs from 28 to 80 µg/cm2. No effect was detected in cells treated with Al2O3 and
ZnO NMs, or the ionic salt control AlCl3. In HepaRG cells, a concentration-dependent increase was also
observed with Al0 NMs from 9 to 80 µg/cm2. In the Fpg-modied comet assay, an increase in tail DNA
was detected in Caco-2 cells treated with Al0 NMs from 9 to 80 µg/cm2 and with Al2O3 NMs at 3, 9 and 28
µg/cm2. Interestingly, in HepaRG cells treated for 5 h with Al NMs, results from the Fpg-modied comet
assay showed that at every concentration tested, only hedgehogs were observed for all NMs (data not
shown).
3.6.3 Interaction of NMs with DNA during the comet assay
The interference of NMs with the comet assay was assessed (Figure 8) according to the protocol
described by Bessa et al [35]. Compared to the untreated control, a concentration-dependent increase in %
tail DNA was observed when Al0 NMs are added at nal concentrations of 9 and 40 µg/cm2. A similar
effect was also observed when Fpg was included in the assay. Compared to the negative control, no
difference was detected for Al2O3 NMs in the absence of Fpg, while a slight increase was observed with
Fpg.
3.6.4 Micronucleus assay
In order to evaluate chromosome damage, the cytokinesis-block micronucleus assay was performed in
Caco-2 and HepaRG cells treated for 24 h (Table 4) with Al NMs. No modication of cell viability (RI
value) was observed in either Caco-2 or HepaRG cells exposed to Al0, Al2O3, ZnO NMs and AlCl3.
Compared to the negative control, no signicant increase in the percentage of BNMN cells was detected
Page 14/39
in either cell line. Similarly, no increase in micronucleated mononuleated cells or in polyploid cells was
observed (data not shown).
4. Bhas-42 Cell transforming (CTA) assay
Results of Bhas-42 CTA performed with Al0 and Al2O3 NMs and with AlC13 are shown in Table 5. In the
initiation assay, both Al0 NMs and AlC13 induced a concentration-dependent decrease in cell proliferation
on Day 7, inhibiting around 90% of cell proliferation at the highest concentration (3 µg/cm2 for Al0 NMs
and 28 µg/ml for AlCl3). In contrast, Al2O3 NMs in the initiation assay and the three Al forms in the
promotion assay induced no, or only a moderate, decrease in cell proliferation for all concentrations
tested. No transforming activity was shown with the three Al forms, irrespective of the concentration
tested in both the initiation and promotion assays. In contrast, the number of foci was found lower than
those of controls at some concentrations of Al and Al2O3.
4. Discussion
Exposure of the general population to NMs present in consumer products, including food, has increased
dramatically within the last decade, and a thorough evaluation of the potential adverse effects resulting
from exposure to NMs following ingestion is necessary. Among the toxic effects of Al-containing NMs
that have been shown in several studies, genetic damage is of particular concern [15, 21, 22, 23]. Both
intestine and liver are considered key organs for investigating genotoxic effect of nanomaterials found in
food since they represent the main organ of contact and the main organ of accumulation, respectively.
Nevertheless, in our recent
in vivo
study investigating the genotoxicity of Al NMs, only a very limited
genotoxic response was observed. In fact, only a cross-linking effect was suggested in the rat duodenum
with Al0 NMs [16]. As the
in vivo
treatment duration was rather short (3 administrations over 2 days), and
that it cannot be excluded that the level of NMs in the organs would be low, we chose to complete our
study by investigating the
in vitro
genotoxicity of Al NMs in human intestinal Caco-2 and hepatic HepaRG
cells using complementary tests.
Despite the uptake and presence of Al NMs in Caco-2 and HepaRG cells, no cytotoxicity or apoptotic
response was observed following treatment with Al2O3 NMs. Our results are in agreement with data from
various publications that have reported little or no cytotoxicity in various cell lines [21, 27, 34, 50, 51],
including in Caco-2 cells [52, 53] and HepG2 cells [22].
No induction of chromosomal damage was observed in the micronucleus assay in either Caco-2 or
HepaRG cells exposed to Al2O3 NMs. Moreover, we did not observe a transforming activity in the CBA
assay, supporting the absence of mutagenic potential for Al2O3 NMs. Our results are consistent with two
recent studies that reported a negative response in the chromosomal aberration and the micronucleus
assays in human lymphocytes treated with Al2O3 NMs with a smaller size (3 to 4 nm) than the one used
in this study (20 nm), and for a longer incubation time (72 h) [20, 54]. In contrast, other studies have
Page 15/39
reported an increase in micronucleus formation following a 24 h treatment with Al2O3 NMs in other cell
lines, including CHO cells [23], human broblasts [21] and RAW264 murine macrophages [24].
Interestingly, Al2O3 NMs were shown to inhibit the replication eciency of high-delity DNA polymerase
[55]. Nevertheless, such inhibition did not affect the mutation rate at the single nucleotide level of
replication products compared to controls [55]. Further investigation demonstrated that Al2O3 NMs did
not induce a clastogenic effect but rather chromosome loss and polyploidy, although these effects were
observed only at one concentration [21]. An aneugenic effect of Al NMs was not observed in our study
(data not shown). The discrepancy may be explained by the fact that our tests were performed in non-
proliferating cells.
Similarly, numerical chromosomal damage (aneuploidy and polyploidy) and abnormal metaphases were
reported in the bone marrow of rats 48 hours after a single oral dose of Al2O3 NMs while no effect was
observed with bulk Al2O3 [14, 15]. In addition, induction of micronuclei in erythrocytes was also observed.
However, this genotoxic effect on erythrocytes was concomitant with a cytotoxic effect, while no toxicity
was observed in our study [16], or in the study of Zhang et al [56]. In contrast, other results obtained from
in vivo
studies are in agreement with the lack of chromosomal damage observed
in vitro
in our study
following treatment with Al2O3 NMs. In fact, with the same Al2O3 NMs used in this study, we did not
observe an induction of micronuclei in either bone marrow or in the colon of rats after a short-term oral
treatment [16]. Similarly, no induction of micronuclei in the bone marrow of mice was detected following
intraperitoneal injections, irrespective of the size of the Al2O3 particles [56].
The absence of genotoxic activity of Al2O3 NMs in Caco-2 and HepaRG cells was further conrmed in the
H2AX assay as well as the comet assay. We did not observe any increase in H2AX levels in either cell
line, which is in agreement with results from a study by Tsaousi et al [21] in primary human broblasts.
Additionally, Al2O3 NMs did not induce DNA damage in the alkaline comet assay in Caco-2 and HepaRG
cells following a 24 h treatment. Although some studies have reported negative results in the comet
assay in human lymphocytes and in human embryonic kidney cells [20, 26], others have demonstrated
time- and/or concentration-dependent genotoxic effects in Chinese hamster lung broblasts [56], in
RAW264 murine macrophages [24] and in human liver HepG2 cells [22] treated with Al2O3 NMs.
Nevertheless, the increase of DNA fragmentation in these latter studies was probably linked to cell death
detected by Trypan blue exclusion [56] or by apoptotic markers [22, 24].
In vivo
, after a short-term treatment using the same Al2O3 NMs, we only observed an increase in DNA
damage in the comet assay in bone marrow, while no effect was observed in intestine, colon, kidney,
spleen or blood [16]. Balasubramanyam et al [14] showed a time- and concentration-dependent increase
in DNA damage in blood with the comet assay with both bulk and nano Al2O3 forms after a single gavage
but the effect decreased at 48 h before disappearing at 72 h. DNA breakage associated with necrosis
and apoptosis was observed in liver and kidney of rats after a repeated oral treatment for 75 days with 70
mg/kg bw Al2O3 NMs [57]. Therefore, it seems that both the
in vitro
and
in vivo
results with Al2O3 NMs
Page 16/39
support the conclusion that DNA breaks detected by the comet assay were mostly related to cell death
rather than to a clear genotoxicity.
Nevertheless, we have shown that Al2O3 NMs induced oxidative DNA damage in Caco-2 cells following a
24 h treatment, despite no signicant ROS induction. Furthermore, a concentration-dependent trend
towards oxidative damage was observed at 5 h. This could suggest the rapid formation of oxidative DNA
damage which is further repaired, as previously demonstrated [58, 59]. Evidence from
in vitro
experiments
in a variety of different cell lines suggests that treatment with Al2O3 NMs can induce oxidative stress [20,
56, 60, 61] including in Caco2 cells [53]. Interestingly, Alari et al [22] reported positive results in the comet
assay in HepG2 cells which was accompanied by oxidative damage and cell death. In the present study,
no oxidative DNA damage or oxidative stress was observed in HepaRG cells. Differentiated HepaRG cells
represent a model which is more similar to human hepatocytes when compared to HepG2 cells, and could
therefore be less sensitive to oxidative damage resulting from Al2O3 NMs. Similarly, we did not detect
oxidative DNA damage in liver, or in other organs of rats after oral exposure [16]. In contrast, an increase
in oxidative stress was observed in several tissues including liver after acute and repeated oral exposure
of rats with Al2O3 NMs [33].
Similar to the results obtained for Al2O3 NMs, no cytotoxicity or apoptotic response was observed
following treatment with Al0 NMs, despite their presence in the cytoplasm of Caco-2 and HepaRG cells. In
contrast to our results in differentiated Caco-2 and HepaRG cells, Al0 NMs were found to induce a
decrease in viability in rat alveolar macrophages and in BRL3A rat liver cells following 24 h exposure at
concentrations similar to those used in our study [27, 62]. This discrepancy could be explained by a
difference in relative cell density for a similar concentration of Al0 NMs tested with a lower NM:cell ratio
in differentiated Caco2 and HepaRG cells compared to the two other proliferating cell systems.
Despite only a slight increase in H2AX levels observed only in HepaRG cells and only at the highest
concentration tested, a dose-dependent increase in tail DNA was observed in both Caco-2 and HepaRG
cell lines treated with Al0 NMs using the alkaline comet assay after both 5 h and 24 h treatments.
Nevertheless, this result required further investigation due to possible interference of NMs with the
alkaline comet assay that has been widely documented in the literature [35, 36, 63, 64]. Indeed, NMs
present in the cytoplasm of cells following uptake can interact with DNA following the lysis step of the
comet assay, and could therefore induce additional breaks or inhibit DNA migration. In addition, a
dissolution due to the conditions of the comet assay could result in reaction of aluminum ions with DNA,
especially the phosphate backbone, as reported in some studies [65, 66]. Such reactions may then induce
DNA damage revealed during the comet assay as suggested by Zhang et al [67]. Our results clearly
demonstrate that, unlike Al2O3 NMs, Al0 NMs can induce DNA damage when in contact with DNA and
interfere signicantly with the comet assay. Consequently, the positive results in cells treated with Al0
NMs obtained in this study should therefore be treated with caution.
In vivo
, using the same Al0 NMs as
the present study, no genotoxic response was observed in several key tissues, with the exception in rat
duodenum where a cross-linking effect was suggested [16].
Page 17/39
The carcinogenic potential of Al NMs was investigated using the cell transformation assay with Bhas 42
cells. Neither Al0 nor Al2O3 NMs induced cell transformation, although a decrease in the number of
transformed foci was observed. This decrease, observed at concentrations inducing a weak inhibition of
cell proliferation at Day 7, is likely explained by a more pronounced inhibition of cell growth after 21 days
of culture due to the three repeated treatments during the promotion assay. This phenomena was also
observed with amorphous silica NMs [49] as well as with other non-carcinogenic chemicals such as L-
ascorbic acid and caffeine [68].
Ion release from NMs in cell culture media, or in intracellular compartments can contribute to cytotoxic
effects
in vitro
. The soluble fraction of Al0 and Al2O3 NMs measured by ICP-MS demonstrated a very low
solubility of Al0 and Al2O3 NMs in both cell media . However, ion release may occur after cell uptake in
specic compartments with low pH such as lysosomes [69] as suggested for Al2O3 NMs [24]. In such a
scenario, secondary effects affecting mitochondria and resulting in the generation of ROS cannot be
excluded. In the case of Al0, the formation of a passivating oxide layer may inuence its dissolution
behavior [70]. Consequently, effects could be induced by ionic Al released from the NMs rather than
effects related to the particulate form [1]. As a strong oxygen acceptor, the Al ion tends to bind to citrate,
phosphate, and catecholamine, generating oxygen radicals [1, 71]. In addition, Al ions can also bind to
negatively charged phospholipids, which are easily attacked by reactive oxygen species such as O2·,
H2O2, and OH· [72, 73] as well as DNA [66].
No genotoxic effects were observed in differentiated Caco-2 or HepaRG cells treated with AlCl3 at
concentrations up to 128 µg/mL Al content corresponding to 1.16 mg/mL AlCl3. At the concentrations of
AlCl3 tested, no effects were observed in the different assays following 5 or 24 h treatments. Indeed,
negative results were obtained for promotion and initiation, as well as for genotoxic and oxidative stress
responses. Our results are consistent with Villarini et al [74] who observed no genotoxicity in response to
Al ions in neuroblastoma cells with the comet assay, as well as no cytotoxicity or oxidative stress.
However, other studies have reported genotoxicity of AlCl3 in human lymphocytes [17, 29]. Interestingly,
the authors of this study observed the highest level of micronuclei during the G1-phase of the cell cycle.
The differentiated HepaRG and Caco2 cells used in our study are not proliferating, and therefore could
explain the discrepancy between the studies.
In vitro
, chromosomal damage observed in blood cells at
AlCl3 concentrations below 25 µg/mL, was associated with apoptosis [28, 29, 30]. Moreover it was shown
that Al ions can induce oxidative DNA damage irrespective of the cell cycle phase [29]. Indeed, the role of
Al ions in mediating genotoxic effects may be more complex, as it has been suggested that Al ions may
inhibit several DNA repair proteins with zinc nger domains [29, 75].
In our study, as the soluble fraction of AlCl3 was always higher than that for Al0 and Al2O3 NMs, the
effects observed for Al0 and Al2O3 NMs are not likely to be related to ion release in cell media. Although
ECHA emphasized that the difference in the toxicological prole between soluble aluminum compounds
and insoluble aluminum oxide may be explained by lower bioavailability of insoluble test compounds, it
Page 18/39
was recently shown that the content of Al in blood of rats treated orally was higher with Al2O3 NMs than
with AlCl3 [76]. Moreover, the persistence of NMs in organs long after intial exposures has been described,
and the accumulation of Al NMs in organs following repeated exposure could poteniate adverse effects
in tissues in the long term. Further studies are clearly needed to investigate the fate of accumulated NMs
in tissue, including possible effects due to ion release, as well as toxic effects related to particle
accumulation.
5. Conclusion
In summary, despite the uptake and presence of Al NMs in the cytoplasm of differentiated Caco-2 and
HepaRG cells, we have shown that Al2O3 NMs do not induce apoptosis, oxidative stress, or cytotoxic
effects following a 24 h treatment. In addition, Al2O3 NMs were negative in the micronucleus assay, and
in initiation and promotion in the CTA. Nevertheless, oxidative DNA damage was observed in Caco-2 cells.
The assays performed with Al0 NMs and AlCl3 were also negative except a slight increase of H2AX levels
only in HepaRG cells, and only at the highest concentration tested. Considerable DNA damage was
observed with Al0 NMs in both Caco-2 and HepaRG cells in the comet assay, although this was likely
associated with the signicant interference with these NMs, and these results must be taken with caution.
As ion release from Al NMs was shown to be very limited in cell media, the effects are rather due to the
particulate form or to ion release inside the cells. Further investigation is needed to clarify the extent of
intracellular ion release from NMs, its contribution to cytotoxic effects compared to the direct impact of
the presence of intracellular particles.
Declarations
Ethical Approval and Consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of supporting data
The datasets used and/or analysed during the current study are available from the corresponding author
on reasonable request.
Competing interests
Page 19/39
The authors declare that they have no competing interests.
Funding
This publication arises from the French-German bilateral project SolNanoTOX funded by the German
Research Foundation (DFG, Project ID: DFG (FKZ LA 3411/1-1 respectively LA 1177/9-1) and the French
National Research Agency (ANR, Project ID: ANR-13-IS10-0005).
Authors' contributions
PJ and AB performed the electron microscopy study and analysis. BCK performed the experiments for
dispersion and dissolution characterization. CF and YG performed and analysed the CTA. FG and SC
performed the density and dispersion chracterisation. PJ, SH and KH performed the genotoxicity
experiments. PJ, BCK, FG, YG, KH and VF wrote the manuscript. AB, FG, AlL, PL, AnL, KH and VF wrote the
proposal to obtain funding. All authors read and approved the nal manuscript.
Acknowledgements
The authors would like to thank Rachelle Lanceleur and Marie-Thérèse Lavault for technical assistance.
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Tables
Table 1: Summary of NM characteristics as reported by the supplier.
Page 26/39
NM NM-code Average particle
sizea
(nm)
SSAb
(m2/g)
Purity
cBulk density
True
densityd
(g/cm3)
Morphology
Al0 NM-0015-
HP 18 40-60 > 99% 2.70
0.008-0.20
spherical
Al2O3 NM-0036-
HP 20 <200 99% -
0.9
spherical
ZnO NM-0011-
HP 20 50 99.5% 5.6
0.3-0.45
Nearly
spherical
a Average particle size was determined by TEM
b Average specic surface area (SSA) was determined by Brunauer-Emmet-Teller (BET)
c Purity was determined by X-ray Powder Diffraction (XRD)
d Density was assessed by normal volumetric test
Table 2: Physico-chemical characterization of Al0, Al2O3 and ZnO NMs.
Page 27/39
Sample
(100 µg/ml)
PdI Z-Ave (d.nm) PdI Z-Ave (d.nm)
Dispersion solution (0h) Dispersion solution (24 h)
Al0 0.173 ± 0.004 254 ± 4 0.159 ± 0.026 253 ± 12
Al2O3 0.235 ± 0.015 168 ± 3 0.186 ± 0.021 160 ± 2
ZnO 0.104 ± 0.038 233 ± 11 0.112 ± 0.045 189 ± 18
DMEM 10% FBS (0h) DMEM 10% FBS (24h)
Al0 0.176 ± 0.011 197 ± 2 0.156 ± 0.011 201 ± 1
Al2O3 0.521 ± 0.027 81 ± 1 0.337 ± 0.041 108 ± 1
ZnO 0.262 ± 0.010 198 ± 4 0.178 ± 0.019 156 ± 9
William’s 5% FBS (0h) William’s 5% FBS (24h)
Al0 0.158 ± 0.008 240 ± 14 0.152 ± 0.007 246 ± 12
Al2O3 0.466 ± 0.015 107 ± 2 0.442 ± 0.018 120 ± 2
ZnO 0.233 ± 0.030 208 ± 6 0.165 ± 0.011 182 ± 11
The mean hydrodynamic diameter (z-Ave) and polydispersity index (PdI) were determined in the stock
dispersion solution and cell media (DMEM + 10 % FBS and William’s + 5 % FBS) after 0 h and 24 h at a
concentration of 100 µg/ml. Three independent experiments were performed. Results are expressed as
mean ± SD.
Table 3: Ion release from Al0, Al2O3, ZnO NMs and AlCl3.
Page 28/39
NMs NM concentration
(µg/mL) Dispersion stock solution
[%] DMEM + 10% FBS
[%] William’s +
5% FBS [%]
Al0 25 1.30 ± 0.06 3.88 ± 0.13 2.42 ± 0.07
50 0.85 ± 0.05 1.94 ± 0.09 1.26 ± 0.07
100 0.48 ± 0.02 0.95 ± 0.04 0.68 ± 0.01
Al2O3 25 0.24 ± 0.04 0.40 ± 0.03 0.32 ± 0.11
50 0.18 ± 0.08 0.48 ± 0.06 0.35 ± 0.07
100 0.15 ± 0.01 0.37 ± 0.02 0.27 ± 0.02
AlCl3 25 89.60 ± 5.97 57.16 ± 5.97 23.35 ±
0.26
50 81.72 ± 0.77 30.09 ± 0.44 12.86 ±
0.28
100 94.31 ± 6.61 17.54 ± 0.72 6.47 ± 0.52
ZnO 25 29.21 ± 0.53 85.09 ± 5.30 54.99 ±
1.03
50 15.07 ± 0.10 57.97 ± 0.68 28.54 ±
1.18
100 7.40 ± 0.05 30.21 ± 0.23 14.74 ±
0.21
Ion release was determined by ICP-MS in the stock dispersion solution and the cell media (DMEM + 10 %
FBS and William’s + 5 % FBS) after 24 h at concentrations of 25, 50 or 100 µg/mL. Data are presented as
the mean ± SD of three independent experiments.
Table 4: Detection of chromosomal damage in differentiated Caco-2 and HepaRG cells treated with Al-
containing NMs.
Page 29/39
Caco2 HepaRG
%BNMN RI (%) % BNMN RI (%)
Control 0 5.6 ± 0.3 100 ± 0 3.6 ± 0.5 100 ± 0
Al0 NMs
[µg/cm2]
3 5.7 ± 0.4 97 ± 0.8 3.2 ± 0.3 97 ± 5.7
9 5.7 ± 0.5 99 ± 3 3.2 ± 0.2 99 ± 3.1
28 5.5 ± 0.4 103 ± 3.3 2.4 ± 0.2 101 ± 6.7
40 5.9 ± 0.6 98 ± 2.6 3.5 ± 0.9 101 ± 5.5
80 6 ± 0.5 94 ± 0.6 2.2 ± 0.3 110 ± 4.5
Al2O3 NMs
[µg/cm2]
3 6.4 ± 1 102 ± 4 3.6 ± 0.6 99 ± 3.3
9 5.6 ± 1.4 100 ± 2.9 3.3 ± 0.3 102 ± 5.2
28 6.3 ± 1.4 96 ± 2.2 4.5 ± 0.8 109 ± 7.2
40 6.4 ± 0.7 98 ± 0.7 3.5 ± 0.5 106 ± 6.8
80 6.4 ± 1 102 ± 4 3.6 ± 0.6 99 ± 3.3
AlCl3
[µg/ml]
28 6.9 ± 0.5 103 ± 1.5 4.7 ± 1.5 114 ± 3.8
40 6.9 ± 0.8 100 ± 1 4.3 ± 0.8 113 ± 3.3
ZnO NMs
[µg/cm2]
3 7 ± 0.8 100 ± 4.1 3.7 ± 0.4 96 ± 3.5
6 8 ± 0.7 96 ± 3.8 4.1 ± 0.4 90 ± 3.4
MMS [µg/mL] 12.4 ± 0.6 ** 39 ± 5.7 ** 11.2 ± 2.4 ** 96 ± 4.9
Cells were treated with increasing concentrations of Al0, Al2O3 and ZnO NMs, and with the ionic salt
control AlCl3. MMS was used as a positive control (25 µg/ml for Caco-2 cells and 30 µg/ml for HepaRG
cells). Results are presented as means (±SEM) of the percentage of binucleated micronucleated cells
(BNMN) scored from 1000 binucleated cells per slide. Two slides per concentration were scored per
experiment. Viability was calculated by the replicative index (RI). Each concentration was tested in
duplicate,
n
= 3. The percentages of BNMN cells were compared using the one-way Pearson chi-square
test.***p<0.01.
Table 5: Effects of Al-containing NMs on cell growth and foci numbers in the CTA assay in Bhas-42 cells.
Cells were treated from day 1 to 4 (initiation assay) or from day 4 to 14 (promotion assay) with Al0 and
Al2O3 NMs, and with AlC13 and post-cultivated in fresh medium until Day 21. MCA (1 µg/mL) and TPA
(0.05 µg/ml) were included as positive controls. The mean of the cell growth (CG) and the number of
Page 30/39
transformed foci per well (foci/well) were measured from 3 and 6 replicate cultures respectively,
according to the OECD.
Page 31/39
Initiation assay Promotion assay
Chemical Concentration CG a Foci/well b CG a Foci/well b
Al 0 c (0.005% BSA) 100 5.3±1.5 100 8.7±3.1
0.03 μg/cm2 107 6.5±1.4 99 4.7±2.1 *
0.1 μg/cm2 105 5.3±2.4 97 0.7±0.8 *
0.3 μg/cm2 96 4.2±0.8 89 0.7±0.8 *
1 μg/cm2 62 2.7±1.6 * 83 Toxic d
3 μg/cm2 14 1.5±0.8 * 75 Toxic d
Al2O3 0 c (0.005% BSA) 100 5.3±1.5 100 8.7±3.1
0.3 μg/cm2 106 3.0±2.0 * 103 5.7±2.0 *
1 μg/cm2 94 3.3±1.5 * 95 7.0±1.3
3 μg/cm2 97 3.8±0.8 93 5.8±1.6 *
9 μg/cm2 74 3.5±1.9 83 4.0±1.5 *
28 μg/cm2 72 2.3±1.6 * 84 0.7±0.5 *
AlCl3 0 c (2.5% H2O) 100 4.7 ± 1.6 100 6.8 ±1. 7
3 μg/ml 96 4.7±1.0 96 Toxic d
9 μg/ml 78 4.3±2.4 88 Toxic d
28 μg/ml 16 4.0±1.3 80 Toxic d
MCA 0 c (0.1% DMSO) 100 5.8±1.9
1 µg/ml 17 14.7±2.9 ≠
TPA 0 c (0.1% DMSO) 100 8.8±2.1
0.05 µg/ml 112 20.3 ±3.0 ≠
Page 32/39
a % of cell growth compared to that of vehicle control.
b Average number of transformed foci/well±SD.
c Vehicle control: nal vehicle concentration of the working culture media in parentheses.
d Absence of cells in well.
*
p
<0.05; Dunnett test,
vs
vehicle control.
≠
p
<0.05;
t
-test,
vs
DMSO (the vehicle of MCA and TPA).
Figures
Figure 1
Page 33/39
Time evolution over 24 h of the well-bottom concentration of (a) Al0 NMs in DMEM; (b) Al2O3 NMs in
DMEM; (c) Al0 NMs in William’s medium; (d) Al2O3 NMs in William’s medium. Model parameters : bulk
NM concentration 250 µg.mL-1; T=37°C.
Figure 2
TEM images of differentiated Caco-2 cross-sections showing the uptake of Al0 NMs (B, C) at 21 µg/cm2
and Al2O3 NMs (D,E) at 39 µg/cm2, after 24 h treatment compared to the negative control (A).
Concentrations of 21 µg/cm2 Al0 NMs and 39 µg/cm2 Al2O3 NMs were used to ensure equivalent Al
content per well. C and E represent enlarged pictures from B and D respectively. C: Al0 NMs were rarely
Page 34/39
seen in proximity to the nucleus (full arrow). C, E: NMs are present either in small clusters in the
cytoplasm or near the nucleus (open arrow) either as large agglomerates integrated into lucent or dense
vesicles (notched arrows). Scale bar 1µm, C: Cytoplasm, N: nucleus.
Figure 3
TEM images of differentiated HepaRG cross sections showing the uptake of Al0 (B, C) at 21 µg/cm2 and
Al2O3 (D,E) at 39 µg/cm2, after 24 h cell treatment compared to the negative control (A). Concentrations
of 21 µg/cm2 Al0 NMs and 39 µg/cm2 Al2O3 NMs were used to ensure equivalent Al content per well. C
and E represent enlarged pictures from B and D respectively. C: Al0 NMs were rarely seen near the nucleus
Page 35/39
(full arrow). E: NMs are included in vesicles containing large agglomerates (notched arrows) or as dense
vesicles containing small clusters in the cytoplasm (open arrow) or as isolated clusters. Scale bar 1µm, C:
Cytoplasm, N: nucleus.
Figure 4
Effects of Al-containing NMs on cell numbers and active caspase-3 levels in differentiated Caco-2 and
HepaRG cells. Cells were treated for 24 h with Al0 NMs, Al2O3 NMs and AlCl3 as ionic salt control. ZnO
and DTX-2 (20 nM for Caco-2 cells, 15 nM for HepaRG) were used as positive controls. Cell numbers (A)
and active-caspase-3 (B) from automated image analysis are presented as fold changes relative to
untreated cells. Representative images of active caspase-3 (green) in the cytoplasm of HepaRG cells are
shown. C) negative control, D) Al0 NMs 80 µg/cm2, E) Al2O3 NMs 80 µg/cm2, F) AlCl3 128 µg/ml, G)
ZnO NMs 6 µg/cm2. Data are presented as the means ± SEM of 3 (Caco-2) or 4 (HepaRG) independent
experiments. ****p< 0.0001. White bar = 100 µm
Page 36/39
Figure 5
Effects of Al-containing NMs on ROS levels in differentiated Caco-2 and HepaRG cells. A) Cells were
treated for 24 h with Al0 NMs, Al2O3 NMs and AlCl3 as ionic salt control. ZnO and Menadione (MEN) (25
µM for HepaRG, 50 µM for Caco-2 cells) were used as positive controls. Representative images of ROS
detection (yellow) in the cytoplasm of HepaRG cells are shown. B) negative control, C) Al0 NMs 80
µg/cm2, D) Al2O3 NMs 80 µg/cm2, E) AlCl3 (128 µg/ml), F) ZnO NMs 6 µg/cm2. Data are presented as
the means ± SEM of 5 (Caco-2) or 6 (HepaRG) independent experiments. *p< 0.05, ***p<0.001. White bar
= 100 μm.
Page 37/39
Figure 6
Effects of Al-containing NMs on γH2AX level in Caco-2 and HepaRG Cells. A) Cells were treated for 24 h
with Al0 NMs, Al2O3 NMs and AlCl3 as ionic salt control, or positive controls (MMS at 60 or 30 µg/ml
respectively, and ZnO NMs). Representative images of γH2AX detection (red) in the nuclei of HepaRG
cells: B) negative control, C) Al0 NMs 80 µg/cm2, D) Al2O3 NMs 80 µg/cm2, E) AlCl3 128 µg/ml, F) ZnO
NMs 6 µg/cm2. Data are presented as the mean ± SEM of 3 independent experiments. *p< 0.05,
***p<0.001, ****p<0.0001. White bar=100 µm.
Page 38/39
Figure 7
Detection of DNA damage in differentiated Caco-2 and HepaRG cells treated 24 h with Al-containing NMs
with the alkaline and Fpg-modied comet assays. DNA damage (A, B) and oxidative DNA damage (C, D)
were assessed in differentiated Caco-2 and HepaRG cells treated for 24 h with Al0, Al2O3 and ZnO NMs,
and with the ionic salt control AlCl3. MMS was used as a positive control (30 µg/ml). Values are
presented as the mean percentage ± SEM of 3 independent experiments. *p < 0.05, **p < 0.01, ***p<0.001,
****p<0.0001.
Page 39/39
Figure 8
Interference of Al0 and Al2O3 NMs with the alkaline and Fpg-modied comet assays. The interference of
Al0 and Al2O3 NMs with DNA migration was assessed with untreated HepaRG cells. NMs were added in
LMP when cells are deposited on slides and compared with control (cells without addition of NMs. Values
are presented as the mean percentage ± SEM of 2 independent experiments.
Supplementary Files
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SuppdatPFT.ppt
