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 modified 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 scientific (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)).
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 final 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 first measured the volume of the potentially agglomerated NM in DMEM and Williams media, at a NM concentration of 250 µg.mL-1, using a specific 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 Analysis device (Malvern Instrument) by measuring the apparent hydrodynamic radius of 400 nm 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 : 0.250 mg.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 16,000 g for 1 h 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 Scientific GmbH, Dreieich, Germany) equipped with a PFA ST Nebulizer, a quartz cyclonic spray chamber and a 2.5 mm quartz injector (Thermo Fisher Scientific). The gas flows were set to 14 L/min, and 0.65 L/min for the cool gas (Ar) and the auxiliary gas (Ar) respectively. The flow 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 fixed 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 Scientific, Waltham, USA) as described in [40]. Cell numbers were determined from DAPI staining, active caspase-3 was quantified in the total cell compartment and H2AX in cell nuclei.
Oxidative stress was measured using CellROX Deep Red Reagent (Fisher Scientific, Illkirch, France). Briefly, 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 fields (20 × magnification) were analyzed for quantification of fluorescence at 647 nm.
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 modified 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]. Briefly, dilutions of Al0 or Al2O3 NMs in 0.5% low-melting point agarose (LMP) were prepared at final 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 guidance document produced by the OECD [48], with some modifications. 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 fixed 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 fixed 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 significance 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 significance of the positive controls (MCA and TPA) was evaluated relative to the control (p < 0.05) by the one-sided Student's t-test.