Pégah Jalili

Sylvie Huet

Agnès Burel

Benjamin-Christoph Krause

Caroline Fontana

Soizic Chevance

Fabienne Gauffre

Yves Guichard

Alfonso Lampen

Peter Laux

Andreas Luch

Kevin Hogeveen

Valérie Fessard
email: valerie.fessard@anses.fr

France ) Methyl

Genotoxic impact of aluminum-containing nanomaterials in human intestinal and hepatic cells

Keywords: nanomaterials, aluminum, nanotoxicology, comet assay, micronucleus, cell transformation assay

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 [START_REF] Li | Aluminum hydroxide nanoparticles show a stronger vaccine adjuvant activity than traditional aluminum hydroxide microparticles[END_REF][START_REF] Shepard | An Occupational Exposure Assessment for Engineered Nanoparticles Used in Semiconductor Fabrication[END_REF][START_REF] Som | Environmental and health effects of nanomaterials in nanotextiles and façade coatings[END_REF][START_REF] Willhite | Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts[END_REF]. Forms of Al, both in the micro-and the nano-size, are present in food and consumer products [START_REF] Saiyed | Aluminium content of some foods and food products in the USA, with aluminium food additives[END_REF][START_REF] Willhite | Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts[END_REF] due to their use as firming, anticaking, neutralizing, emulsifying and texturizing agents, as well as for cooking tools [START_REF] Moradi | Development and characterization of kefiran -Al2O3 nanocomposite films: Morphological, physical and mechanical properties[END_REF], waste water treatment (European Food Safety, 2008;[START_REF] Kumar | Defluoridation from aqueous solutions by nano-alumina: Characterization and sorption studies[END_REF] and in medical and hygiene products such as toothpaste [START_REF] Narayan | Atomic layer deposition-based functionalization of materials for medical and environmental health applications[END_REF]Zhang et al., 2017a;[START_REF] Zhao | Toxicology of nanomaterials used in nanomedicine[END_REF]). Nevertheless, their potential toxicity has not been fully evaluated, leading to major concerns from consumers and public health agencies [START_REF] Laux | Nanomaterials: certain aspects of application, risk assessment and risk communication[END_REF].

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 (TWI) (1 mg/kg bw/week) (European Food Safety, 2008). In addition, a recent study has estimated total consumer exposure to Al containing compounds, including contributions from products used in food (additives, contact materials) and in cosmetics, and concluded that most of the classes exceeded the EFSA-derived TWI, and that adolescents were highly exposed [START_REF] Tietz | Aggregated aluminium exposure: risk assessment for the general population[END_REF].

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 Al 2 O 3 NMs only.

DNA damage was reported in erythrocytes of rats after a single oral treatment with Al 2 O 3 NMs, although at high doses (≥1,000 mg/kg) (Balasubramanyam et al., 2009a;Balasubramanyam et al., 2009b). Genotoxic effects were observed in bone marrow, but not in other organs, after a short-term treatement with lower doses of Al 2 O 3 NMs [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF].

In vivo effects of Al 0 NMs following oral exposure are mostly lacking, although one study suggested cross-linking effects on DNA in the duodenum of rats [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF].

Following oral exposure of rodents with ionic forms of Al, an increase in micronucleus (MN) frequency was reported in bone marrow after a single oral administration [START_REF] Paz | Evaluation of in vivo and in vitro toxicological and genotoxic potential of aluminum chloride[END_REF] and in liver after a 30 day oral treatment [START_REF] Turkez | Propolis prevents aluminium-induced genetic and hepatic damages in rat liver[END_REF]. Nevertheless, the induction of MN formation in liver was shown to decrease with an antioxidant treatment [START_REF] Turkez | Borax counteracts genotoxicity of aluminum in rat liver[END_REF][START_REF] Turkez | Propolis prevents aluminium-induced genetic and hepatic damages in rat liver[END_REF]. Consistent with these results, a slight oxidative DNA damage was observed in blood after a short-term oral exposure [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF].

The in vitro genotoxicity of Al 2 O 3 NMs has been assessed in several mammalian cell lines including human peripheral lymphocytes [START_REF] Rajiv | Comparative cytotoxicity and genotoxicity of cobalt (II, III) oxide, iron (III) oxide, silicon dioxide, and aluminum oxide nanoparticles on human lymphocytes in vitro[END_REF], primary human fibroblasts [START_REF] Tsaousi | The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles[END_REF], hepatic HepG2 cells [START_REF] Alarifi | Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells[END_REF], and Chinese hamster ovary (CHO) cells [START_REF] Virgilio | Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells[END_REF]. While some studies have not observed genotoxic effects of Al 2 O 3 NMs [START_REF] Demir | Determination of TiO2, ZrO2, and Al2O3 nanoparticles on genotoxic responses in human peripheral blood lymphocytes and cultured embyronic kidney cells[END_REF][START_REF] Hashimoto | Cytotoxic and genotoxic characterization of aluminum and silicon oxide nanoparticles in macrophages[END_REF][START_REF] Mckenna | Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin Cinduced DNA cross-link damage and repair of specific DNA sequences in RT4 cells[END_REF][START_REF] Rajiv | Comparative cytotoxicity and genotoxicity of cobalt (II, III) oxide, iron (III) oxide, silicon dioxide, and aluminum oxide nanoparticles on human lymphocytes in vitro[END_REF], others have reported a positive response [START_REF] Alarifi | Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells[END_REF][START_REF] Tsaousi | The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles[END_REF] which may be associated with oxidative damage [START_REF] Alarifi | Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells[END_REF]. In contrast, data on the in vitro genotoxicity of Al 0 NMs are scarce with no effect reported in the induction of genotoxic markers on human hepatic HepaRG and HepG2 cells [START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF]. For the salt AlCl 3, 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 [START_REF] Banasik | Aluminum-induced micronuclei and apoptosis in human peripheral-blood lymphocytes treated during different phases of the cell cycle[END_REF][START_REF] Lankoff | A comet assay study reveals that aluminium induces DNA damage and inhibits the repair of radiation-induced lesions in human peripheral blood lymphocytes[END_REF][START_REF] Lima | Genotoxic effects of aluminum chloride in cultured human lymphocytes treated in different phases of cell cycle[END_REF][START_REF] Paz | Evaluation of in vivo and in vitro toxicological and genotoxic potential of aluminum chloride[END_REF]. However, no response of the genotoxic markers, phospho-ATM, phospho-p53 and phosphorylated histone H2AX (H2AX), was induced in HepaRG and HepG2 cells [START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF].

According to an European Chemicals Agency (ECHA) safety assessment (ECHA), the data available on the genotoxicity of Al 2 O 3 NMs are inconclusive while few data on the genotoxicity of Al 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 Al 2 O 3 NMs (Balasubramanyam et al., 2009b;[START_REF] Park | A 13-week repeated-dose oral toxicity and bioaccumulation of aluminum oxide nanoparticles in mice[END_REF][START_REF] Shrivastava | Effects of sub-acute exposure to TiO2, ZnO and Al2O3 nanoparticles on oxidative stress and histological changes in mouse liver and brain[END_REF]. Concerning carcinogenicity, although aluminum is not listed as a carcinogen (European Food Safety, 2008), certain exposures occurring during aluminium production cause cancer to humans (1987). Nevertheless, conflicting evidence on Al carcinogenicity has been pointed out. Although no association between Al and breast cancer was recently outlined, additional data on Al toxicity are requested [START_REF] Klotz | The Health Effects of Aluminum Exposure[END_REF][START_REF] Tietz | Aggregated aluminium exposure: risk assessment for the general population[END_REF].

Although we recently investigated the in vivo genotoxicity of Al NMs [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF], the in vivo treatment duration was rather short (3 administrations over 2 days), and it cannot be excluded that the level of NMs in the organs would be low. Therefore, the aim of the current study was to complete the data on the genotoxicity of Al 0 and Al 2 O 3 NMs by evaluating their genotoxic potential in two relevant human cell models of intestine and liver.

Intestinal Caco2 and liver HepaRG cell lines were used as they can differentiate into enterocytes and hepatocyte-like cells, and have been well characterized. These models have been previouslyused for genotoxicity testing with a long standing experience in the Anses laboratory [START_REF] Bazin | Genotoxicity of a freshwater cyanotoxin, cylindrospermopsin, in two human cell lines: Caco-2 and HepaRG[END_REF][START_REF] Hegarat | Assessment of the genotoxic potential of indirect chemical mutagens in HepaRG cells by the comet and the cytokinesisblock micronucleus assays[END_REF][START_REF] Hégarat | Genotoxicity of the marine toxin okadaic acid, in human Caco-2 cells and in mice gut cells[END_REF][START_REF] Hégarat | Performance of comet and micronucleus assays in metabolic competent HepaRG cells to predict in vivo genotoxicity[END_REF]. Several endpoints of genotoxicity were investigated using the alkaline and formamidopyrimidine-DNA glycosylase (Fpg)-modified comet assays which detects DNA breakage including oxidative lesions, DNA double strand breaks were detected through γH2AX, and the MN 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 AlCl 3 . Moreover, the interference of NMs, including with Al-NMs [START_REF] Monteiro-Riviere | Interactions of aluminum nanoparticles with human epidermal keratinocytes[END_REF], has been demonstrated in numerous publications using a wide range of biological assays, and stresses the necessity to evaluate interference in order to assess the potential effect on the results [START_REF] Azqueta | The use of the comet assay for the evaluation of the genotoxicity of nanomaterials[END_REF][START_REF] Bessa | Moving into advanced nanomaterials. Toxicity of rutile TiO(2) nanoparticles immobilized in nanokaolin nanocomposites on HepG2 cell line[END_REF][START_REF] Di Bucchianico | Genotoxicity of TiO2 nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry[END_REF][START_REF] Ferraro | Overestimation of nanoparticles-induced DNA damage determined by the comet assay[END_REF]. In this study, various sources of interference have been taken into account within the different assays.

Materials and methods

Nanomaterials, chemicals and reagents

Al 0 , Al 2 O 3 and zinc oxide (ZnO) NMs with a similar primary particle size were supplied from IoLiTec (Heilbronn, Germany). AlCl 3 (hexahydrate) was purchased from

The human colorectal adenocarcinoma Caco-2 cell line was cultured (passages 25-38) until differentiation after 21 days as described in [START_REF] Jalili | Investigation of the in vitro genotoxicity of two rutile TiO2 nanomaterials in human intestinal and hepatic cells and evaluation of their interference with toxicity assays[END_REF] 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 [START_REF] Aninat | Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells[END_REF][START_REF] Jalili | Investigation of the in vitro genotoxicity of two rutile TiO2 nanomaterials in human intestinal and hepatic cells and evaluation of their interference with toxicity assays[END_REF]. Differentiated Caco-2 and HepaRG cells were treated for 24 h with Al 0 and Al 2 O 3 NMs at concentrations ranging from 0.03 to 80 µg/cm 2 and with AlCl 3 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/cm 2 were used as a positive NM control. Equivalence between volume concentration (µg/mL) and surface concentration (µg/cm 2 ) are shown in

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 [START_REF] Deloid | Preparation, characterization, and in vitro dosimetry of dispersed, engineered nanomaterials[END_REF]. 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:

𝜌 𝑒𝑓𝑓 = 𝜌 𝑚 + [( 𝑀 𝑁𝑃 𝑉 × 𝑆𝐹 ) (1 - 𝜌 𝑚 𝜌 𝑁𝑃 )]
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 Al 2 O 3 ), M NP the total mass of NM in 1 mL of dispended volume and V the measured volume pellet. The stacking factor (SF) 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 [START_REF] Deloid | Preparation, characterization, and in vitro dosimetry of dispersed, engineered nanomaterials[END_REF]. The size of the NMs was taken from Table 2 (Z-Ave). 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).

Ion release from NMs

Following the dispersion of Al 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.

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 [START_REF] Jalili | Investigation of the in vitro genotoxicity of two rutile TiO2 nanomaterials in human intestinal and hepatic cells and evaluation of their interference with toxicity assays[END_REF].

Cellular imaging and High Content Analysis (HCA)

After 24 h treatment with Al NMs and AlCl 3 , plates were processed for HCA with an ArrayScan VTI HCS Reader (Thermo Scientific, Waltham, USA) as described in [START_REF] Jalili | Investigation of the in vitro genotoxicity of two rutile TiO2 nanomaterials in human intestinal and hepatic cells and evaluation of their interference with toxicity assays[END_REF]. 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 phosphate buffered saline (PBS) before treatment with NMs and AlCl 3 . 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. ZnO NMs (6 µg/cm 2 ) and menadione (25 µM for HepaRG and 50 µM for Caco-2 cells) were used as positive controls.

Comet assay

After a 5 h (Figure S 3) or 24 h treatment with Al NMs and AlCl 3 , the comet assay was performed as described in [START_REF] Jalili | Investigation of the in vitro genotoxicity of two rutile TiO2 nanomaterials in human intestinal and hepatic cells and evaluation of their interference with toxicity assays[END_REF][START_REF] Hégarat | A co-culture system of human intestinal Caco-2 cells and lymphoblastoid TK6 cells for investigating the genotoxicity of oral compounds[END_REF]. 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 Journal Pre-proof ACCEPTED MANUSCRIPT / CLEAN COPY damage was high and no clear head of the comet was observed. At least three independent experiments were performed. MMS (30 µg/mL, for 5 and 24h treatments) 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 induced by the excision of oxidized purines [START_REF] Collins | Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA[END_REF][START_REF] Dušinská | Detection of Oxidised Purines and UV-induced Photoproducts in DNA of Single Cells, by Inclusion of Lesion-specific Enzymes in the Comet Assay[END_REF]. 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 2000 U/mL

Fpg at 37°C for 30 min, were then processed as described previously.

Particle interaction with DNA during the comet assay

The interaction of NMs with DNA migration during the comet assay was evaluated as described previously [START_REF] Bessa | Moving into advanced nanomaterials. Toxicity of rutile TiO(2) nanoparticles immobilized in nanokaolin nanocomposites on HepG2 cell line[END_REF][START_REF] Jalili | Investigation of the in vitro genotoxicity of two rutile TiO2 nanomaterials in human intestinal and hepatic cells and evaluation of their interference with toxicity assays[END_REF]. Briefly, dilutions of Al 0 or Al 2 O 3 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/cm 2 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.

Cytokinesis-block micronucleus assay (CBMN)

After a 24h-treatment, the CBMN assay was performed as described in [START_REF] Jalili | Investigation of the in vitro genotoxicity of two rutile TiO2 nanomaterials in human intestinal and hepatic cells and evaluation of their interference with toxicity assays[END_REF] according to the guideline n°487 of the Organization for Economic Co-operation and Journal Pre-proof , 2010). 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 out and each concentration was tested in duplicate. The replication index (RI)

ACCEPTED MANUSCRIPT / CLEAN COPY Development (OECD) (OCDE
was calculated using the formula recommended by OCDE guideline n°487. MMS was used as positive control (25 µg/mL for Caco-2 cells and 30 µg/mL for HepaRG cells).

Bhas 42 Cell Transformation Assay (CTA)

Originally established from the v-Ha-ras-transfected BALB/c 3T3 cells by Sasaki et al [START_REF] Sasaki | Isolation and characterization of ras-transfected BALB/3T3 clone showing morphological transformation by 12-O-tetradecanoyl-phorbol-13acetate[END_REF], 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 (OECD, 2017), with some modifications. The protocol, including both an initiation and a promotion assay, was previously described by Fontana et al [START_REF] Fontana | In vitro cell transformation induced by synthetic amorphous silica nanoparticles[END_REF].

In the initiation assay, 24 h after seeding (420 cells/cm 2 ) (Day 1), the cells were treated with Al NMs and AlCl 3 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/cm 2 ) 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 AlCl 3 . 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 Journal Pre-proof ACCEPTED MANUSCRIPT / CLEAN COPY 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.

Statistical analysis

The statistical significance of HCA and comet assay results was tested using one-way Analysis of variance (ANOVA) followed by Dunnett's post-hoc tests with GraphPad Prism 5.

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 onesided 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.

Results

Nanomaterial characterization

Information concerning the physico-chemical characterization, including the morphology, primary size, surface specific area (SSA), purity and density of the Al 0 , Al 2 O 3 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 Al 2 O 3 NMs in the stock dispersion solution cannot be considered as being spherical, but rather have a rod-like shape Journal Pre-proof when observed by TEM (Figure S1). Although Al 0 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 (Z-Ave) in the stock dispersion solution, as well as in cell media, were assessed by Dynamic Light Scattering (DLS). Measurements were achieved immediately after dispersion (0 h), as well as after 24 h. In both cases, the dispersions were

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 Al 0 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 Al 0 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 Al 0 and ZnO NMs, for Al 2 O 3 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 AlCl 3 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 AlCl 3 in cell media.

Uptake of Al NMs in Caco-2 and HepaRG cells

The uptake and the intracellular distribution of Al Similarly, no significant change in cell numbers were observed in Caco-2 cells treated with ZnO at the concentrations tested. However, a significant decrease in cell numbers as well as an increase in active caspase-3 levels was observed for the highest dose (6 µg/cm 2 ) of ZnO NMs in HepaRG cells. ACCEPTED MANUSCRIPT / CLEAN COPY

Genotoxicity

γH2AX

Quantification 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 Al 0 and Al 2 O 3 NMs up to 80 µg/cm 2 , with ZnO NMs, or with the ionic salt control AlCl 3 up to 128 µg/mL (Figure 6). However in HepaRG cells, Al 0 NMs induced a slight but statistically significant increase in γH2AX levels at the highest concentration (80 µg/cm 2 ) tested. ZnO NMs induced significant increases at 3 and 6 µg/cm 2 .

Comet assay

The potential for Al 0 and Al 2 O 3 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 modified comet assay with the Fpg enzyme was also performed to detect oxidative DNA damage (Figure 7 C and D).

In Caco-2 cells, a significant increase in tail DNA was observed with Al 0 NMs from 28 to 80 µg/cm 2 in the alkaline comet assay (Figure 7 A). In contrast, neither Al 2 O 3 and ZnO NMs, or the ionic salt control AlCl 3 induced any significant increase in tail DNA. In the Fpgmodified comet assay, a significant increase in tail DNA was observed in cells treated with Al 2 O 3 NMs at 3, 9 and 80 µg/cm 2 (Figure 7 C).

In HepaRG cells, tail DNA significantly increased in a dose-dependent manner in cells treated with Al 0 NMs, including a very considerable effect starting at 28 µg/cm 2 . In contrast, no effect was observed for cells treated with Al 2 O 3 and ZnO NMs, or the ionic control AlCl 3 (Figure 7 B). Similarly, an increase in tail DNA in the Fpg-modified comet assay was observed for Al 0 NMs at all concentrations tested with a very significant effect observed at concentrations above 9 µg/cm 2 . No significant changes in tail DNA were detected in HepaRG cells treated with Al 2 O 3 and ZnO NMs or AlCl 3 (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 Al 0 NMs from 28 to 80 µg/cm 2 . No effect was detected in cells treated with Al 2 O 3 and ZnO NMs, or the ionic salt control AlCl 3 . In HepaRG cells, a concentration-dependent increase was also observed with Al 0 NMs from 9 to 80 µg/cm 2 . In the Fpg-modified comet assay, an increase in tail DNA was detected in Caco-2 cells treated with Al 0 NMs from 9 to 80 µg/cm 2 and with Al 2 O 3 NMs at 3, 9 and 28 µg/cm 2 .

Interestingly, in HepaRG cells treated for 5 h with Al NMs, results from the Fpg-modified ACCEPTED MANUSCRIPT / CLEAN COPY comet assay showed that at every concentration tested, only hedgehogs were observed for all NMs (data not shown).

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 [START_REF] Bessa | Moving into advanced nanomaterials. Toxicity of rutile TiO(2) nanoparticles immobilized in nanokaolin nanocomposites on HepG2 cell line[END_REF]. Compared to the untreated control, a concentration-dependent increase in % tail DNA was observed when Al 0 NMs are added at final concentrations of 9 and 40 µg/cm 2 . A similar effect was also observed when Fpg was included in the assay. Compared to the negative control, no difference was detected for Al 2 O 3

NMs in the absence of Fpg, while a slight increase was observed with Fpg.

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

Bhas-42 Cell transforming (CTA) assay

Results of Bhas-42 CTA performed with Al 0 and Al 2 O 3 NMs and with AlC1 3 are shown in Table 5. In the initiation assay, both Al 0 NMs and AlC1 3 induced a concentrationdependent decrease in cell proliferation on Day 7, inhibiting around 90% of cell proliferation at the highest concentration (3 µg/cm 2 for Al 0 NMs and 28 µg/mL for AlCl 3 ). In contrast, Al 2 O 3 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 Al 2 O 3 .

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 [START_REF] Alarifi | Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells[END_REF]Balasubramanyam et al., 2009b;[START_REF] Virgilio | Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells[END_REF][START_REF] Tsaousi | The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles[END_REF]. Both intestine and liver are considered key organs for investigating genotoxic effect of nanomaterials found in food since they represent ACCEPTED MANUSCRIPT / CLEAN COPY the main organ of contact and the main organ of accumulation, respectively. Nevertheless, in our recent in vivo study, only a very limited genotoxic response was observed with Al 0 NMs [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF] while no effect was detected on several genotoxicity markers in vitro [START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF]. Therefore, 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.

Al 2 O 3 NMs

Despite the uptake and presence of Al NMs in Caco-2 and HepaRG cells, no cytotoxicity or apoptotic response was observed following treatment with Al 2 O 3 NMs. Our results are in agreement with data from various publications that have reported little or no cytotoxicity in various cell lines [START_REF] Monteiro-Riviere | Interactions of aluminum nanoparticles with human epidermal keratinocytes[END_REF][START_REF] Radziun | Assessment of the cytotoxicity of aluminium oxide nanoparticles on selected mammalian cells[END_REF][START_REF] Simon-Deckers | In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes[END_REF][START_REF] Tsaousi | The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles[END_REF][START_REF] Wagner | Cellular Interaction of Different Forms of Aluminum Nanoparticles in Rat Alveolar Macrophages[END_REF], including in Caco-2 cells [START_REF] Ivask | Toxicity of 11 Metal Oxide Nanoparticles to Three Mammalian Cell Types <i>In Vitro<[END_REF][START_REF] Sieg | Uptake and molecular impact of aluminumcontaining nanomaterials on human intestinal caco-2 cells[END_REF][START_REF] Song | Comparing Toxicity of Alumina and Zinc Oxide Nanoparticles on the Human Intestinal Epithelium In Vitro Model[END_REF], HepaRG [START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF] and HepG2 cells [START_REF] Alarifi | Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells[END_REF][START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF].

No induction of chromosomal damage was observed in the micronucleus assay in either Caco-2 or HepaRG cells exposed to Al 2 O 3 NMs. Moreover, we did not observe a transforming activity in the CTA assay, supporting the absence of mutagenic potential for Al 2 O 3 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 Al 2 O 3 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) [START_REF] Akbaba | Investigation of the Genotoxicity of Aluminum Oxide, beta-Tricalcium Phosphate, and Zinc Oxide Nanoparticles In Vitro[END_REF][START_REF] Rajiv | Comparative cytotoxicity and genotoxicity of cobalt (II, III) oxide, iron (III) oxide, silicon dioxide, and aluminum oxide nanoparticles on human lymphocytes in vitro[END_REF]. In contrast, other studies have reported an increase in micronucleus formation following a 24 h treatment with Al 2 O 3 NMs in other cell lines, including CHO cells [START_REF] Virgilio | Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells[END_REF], human fibroblasts [START_REF] Tsaousi | The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles[END_REF] and RAW264 murine macrophages [START_REF] Hashimoto | Cytotoxic and genotoxic characterization of aluminum and silicon oxide nanoparticles in macrophages[END_REF]. Interestingly, Al 2 O 3 NMs were shown to inhibit the replication efficiency of high-fidelity DNA polymerase [START_REF] Gao | Impact of metal oxide nanoparticles on in vitro DNA amplification[END_REF], although such inhibition did not affect the mutation rate at the single nucleotide level of replication products compared to controls [START_REF] Gao | Impact of metal oxide nanoparticles on in vitro DNA amplification[END_REF]. Further investigation demonstrated that Al 2 O 3 NMs did not induce a clastogenic effect but rather chromosome loss and polyploidy, although these effects were observed only at one concentration (1 mg/mL) [START_REF] Tsaousi | The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles[END_REF]. Considering the induction of polyploidy, mitosis or increase of micronucleated mononucleated cells, 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 treatments were performed in non-proliferating cells. The lack of chromosomal damage observed in vitro in our study following treatment with Al 2 O 3 NMs is in agreement with results obtained from in vivo studies. In fact, with the same Al 2 O 3 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 [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF]. Similarly, no induction of micronuclei in the bone marrow of mice was detected following intraperitoneal injections, irrespective of the size of the Al 2 O 3 particles (Zhang et al., 2017b). In contrast, 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 Al 2 O 3 NMs while no effect was observed with bulk Al 2 O 3 (Balasubramanyam et al., 2009a;Balasubramanyam et al., 2009b).

In addition, induction of micronuclei in erythrocytes was also observed. However, no toxicity on the bone marrow (determined by the frequency of polychromatic erythrocytes per total erythrocytes) was observed in the different studies (Balasubramanyam et al., 2009a;Balasubramanyam et al., 2009b;[START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF]Zhang et al., 2017b).

The absence of genotoxic activity of Al 2 O 3 NMs in Caco-2 and HepaRG cells was further confirmed 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 [START_REF] Tsaousi | The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles[END_REF] in primary human fibroblasts. Additionally, Al 2 O 3 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 [START_REF] Demir | Determination of TiO2, ZrO2, and Al2O3 nanoparticles on genotoxic responses in human peripheral blood lymphocytes and cultured embyronic kidney cells[END_REF][START_REF] Rajiv | Comparative cytotoxicity and genotoxicity of cobalt (II, III) oxide, iron (III) oxide, silicon dioxide, and aluminum oxide nanoparticles on human lymphocytes in vitro[END_REF], others have demonstrated time-and/or concentration-dependent genotoxic effects in Chinese hamster lung fibroblasts (Zhang et al., 2017b), in RAW264 murine macrophages [START_REF] Hashimoto | Cytotoxic and genotoxic characterization of aluminum and silicon oxide nanoparticles in macrophages[END_REF] and in human liver HepG2 cells [START_REF] Alarifi | Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells[END_REF] treated with Al 2 O 3 NMs. Nevertheless, the increase of DNA fragmentation in these latter studies was probably linked to cell death detected by Trypan blue exclusion (Zhang et al., 2017b) or by apoptotic markers [START_REF] Alarifi | Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells[END_REF][START_REF] Hashimoto | Cytotoxic and genotoxic characterization of aluminum and silicon oxide nanoparticles in macrophages[END_REF].

Moreover, interference of Al 2 O 3 NMs was not checked in these publications which may also explain the discrepancy in the responses.

In vivo, after a short-term treatment using the same Al 2 O 3 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 [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF]. Balasubramanyam et al (Balasubramanyam et al., 2009a) showed a time-and concentration-dependent increase in DNA damage in blood with the comet assay with both bulk and nano Al 2 O 3 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 Al 2 O 3 NMs [START_REF] Yousef | Hepato-renal toxicity of oral sub-chronic exposure to aluminum oxide and/or zinc oxide nanoparticles in rats[END_REF].

Therefore, it seems that both the in vitro and in vivo results with Al 2 O 3 NMs 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 Al 2 O 3 NMs induced oxidative DNA damage in Caco-2 cells following a 24 h treatment, despite no significant ROS induction. Furthermore, a concentration-dependent trend towards oxidative damage was observed at 5 h. In previous studies, we already showed that no oxidative stress (reduced glutathione level and mitochondrial membrane potential) was detected in HepaRG and HepG2 cells as well as on differentiated Caco2 cells after 24h exposure to the same Al 2 O 3 NMs [START_REF] Sieg | Uptake and molecular impact of aluminumcontaining nanomaterials on human intestinal caco-2 cells[END_REF][START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF]. This could suggest the rapid formation of oxidative DNA damage which is further repaired, as previously demonstrated [START_REF] Sadiq | In vitro toxicological assessment of iron oxide, aluminium oxide and copper nanoparticles in prokaryotic and eukaryotic cell types[END_REF][START_REF] Sliwinska | Genotoxicity and cytotoxicity of ZnO and Al2O3 nanoparticles[END_REF].

Evidence from in vitro experiments in a variety of different cell lines suggests that treatment with Al 2 O 3 NMs can induce oxidative stress [START_REF] Li | An acetyl-L-carnitine switch on mitochondrial dysfunction and rescue in the metabolomics study on aluminum oxide nanoparticles[END_REF][START_REF] Rajiv | Comparative cytotoxicity and genotoxicity of cobalt (II, III) oxide, iron (III) oxide, silicon dioxide, and aluminum oxide nanoparticles on human lymphocytes in vitro[END_REF][START_REF] Shah | Nanoscalealumina induces oxidative stress and accelerates amyloid beta (Aβ) production in ICR female mice[END_REF]Zhang et al., 2017b) including in Caco2 cells [START_REF] Song | Comparing Toxicity of Alumina and Zinc Oxide Nanoparticles on the Human Intestinal Epithelium In Vitro Model[END_REF]. Interestingly, Alarifi et al [START_REF] Alarifi | Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells[END_REF] 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 Al 2 O 3 NMs. In support of this, differences in the impact of ionic Al on oxidative stress was observed in HepG2 and HepaRG cells, with a clear reduction of GSH in HepG2 cells whereas no GSH reduction was detected in HepaRG cells [START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF]. Less information on is available concerning the antioxidant capacity of Caco-2 cells, however HepG2 cells were more sensitive than Caco-2 cells to oxidative stress generated by hydrogen peroxide, tertbutylhydroperoxide and 2,2'-azobis (2-amidinopropane) [START_REF] Martin | DIFFERENTIAL SUSCEPTIBILITY OF CACO-2 AND HEPG2 HUMAN CELL LINES TO OXIDATIVE STRESS[END_REF]. The higher antioxidant capacity of the cell lines used in our study could therefore explain the lack of oxidative stress observed following treatment with Al NMs.

Similarly, we did not detect oxidative DNA damage in liver, or in other organs of rats after oral exposure [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF]. In contrast, an increase in oxidative stress was observed in several tissues including liver after acute and repeated oral exposure of rats with Al 2 O 3 NMs [START_REF] Shrivastava | Effects of sub-acute exposure to TiO2, ZnO and Al2O3 nanoparticles on oxidative stress and histological changes in mouse liver and brain[END_REF].

Al 0 NMs

Similar to the results obtained for Al 2 O 3 NMs, no cytotoxicity or apoptotic response was observed following treatment with Al 0 NMs, despite their presence in the cytoplasm of Caco-2 and HepaRG cells, as previously reported [START_REF] Sieg | Uptake and molecular impact of aluminumcontaining nanomaterials on human intestinal caco-2 cells[END_REF][START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF]. In contrast to our results in differentiated Caco-2 and HepaRG cells, Al 0 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 [START_REF] Hussain | In vitro toxicity of nanoparticles in BRL 3A rat liver cells[END_REF][START_REF] Wagner | Cellular Interaction of Different Forms of Aluminum Nanoparticles in Rat Alveolar Macrophages[END_REF]. This discrepancy could be explained by a difference in relative cell density for a similar concentration of Al 0 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 Al 0 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 [START_REF] Azqueta | The use of the comet assay for the evaluation of the genotoxicity of nanomaterials[END_REF][START_REF] Bessa | Moving into advanced nanomaterials. Toxicity of rutile TiO(2) nanoparticles immobilized in nanokaolin nanocomposites on HepG2 cell line[END_REF][START_REF] Karlsson | Can the comet assay be used reliably to detect nanoparticle-induced genotoxicity?[END_REF][START_REF] Magdolenova | Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles[END_REF]. 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 Al ions with DNA, especially the phosphate backbone, as reported in some studies [START_REF] Heli | A study of double stranded DNA adsorption on aluminum surface by means of electrochemical impedance spectroscopy[END_REF][START_REF] Zhang | Spectroscopic and Voltammetric Study on the Binding of Aluminium(III) to DNA[END_REF]. Such reactions may then induce DNA damage revealed during the comet assay as suggested by Zhang et al [START_REF] Zhang | Electrochemical and Spectrometric Studies of Double-Strand Calf Thymus Gland DNA Denatured by Al(III) at Neutral pH[END_REF]. Our results clearly demonstrate that, unlike Al 2 O 3 NMs, Al 0 NMs can induce DNA damage when in contact with DNA and interfere significantly with the comet assay. Consequently, the positive results in cells treated with Al 0 NMs obtained in this study should therefore be treated with caution. In vivo, using the same Al 0 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 [START_REF] Jalili | Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure[END_REF].

The carcinogenic potential of Al NMs was investigated using the cell transformation assay with Bhas 42 cells. Neither Al 0 nor Al 2 O 3 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 [START_REF] Fontana | In vitro cell transformation induced by synthetic amorphous silica nanoparticles[END_REF] as well as with other non-carcinogenic chemicals such as Lascorbic acid and caffeine [START_REF] Sakai | A Bhas 42 cell transformation assay on 98 chemicals: The characteristics and performance for the prediction of chemical ACCEPTED MANUSCRIPT / CLEAN COPY carcinogenicity[END_REF]. However, ion release may occur after cell uptake in specific compartments with low pH such as lysosomes [START_REF] Sabella | A general mechanism for intracellular toxicity of metal-containing nanoparticles[END_REF] as suggested for Al 2 O 3 NMs [START_REF] Hashimoto | Cytotoxic and genotoxic characterization of aluminum and silicon oxide nanoparticles in macrophages[END_REF]. In such a scenario, secondary effects affecting mitochondria and resulting in the generation of ROS cannot be excluded. Consequently, effects could be induced by ionic Al released from the NMs rather than effects related to the particulate form [START_REF] Willhite | Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts[END_REF]. As a strong oxygen acceptor, the Al ion tends to bind to citrate, phosphate, and catecholamine, generating oxygen radicals [START_REF] Harris | Speciation of aluminum in biological systems[END_REF][START_REF] Willhite | Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts[END_REF]. In addition, Al ions can also bind to negatively charged phospholipids, which are easily attacked by reactive oxygen species such as O 2 ·, H 2 O 2 , and OH· [START_REF] Verstraeten | Myelin Is a Preferential Target of Aluminum-Mediated Oxidative Damage[END_REF][START_REF] Verstraeten | Effects of Al3+ and Related Metals on Membrane Phase State and Hydration: Correlation with Lipid Oxidation[END_REF] as well as DNA [START_REF] Zhang | Spectroscopic and Voltammetric Study on the Binding of Aluminium(III) to DNA[END_REF]. Although artificial simulation of the lysosomal compartement was not performed in our study, a previous study demonstrated that these same Al NMs did not dissolve significantly in an in vitro digestion system, even at low pH simulating stomach conditions (pH 2 for 2 hours with specific enzymes). In gastric media, metallic Al 0 nanoparticles released, slightly more ions (3 %), while Al 2 O 3 nanoparticles were more inert in terms of solubility. Such behavior could be explained by the different surface of the two Al NMs as the formation of a passivating oxide layer on Al 0 NMs may influence its dissolution behavior [START_REF] Krause | Characterization of aluminum, aluminum oxide and titanium dioxide nanomaterials ACCEPTED MANUSCRIPT / CLEAN COPY using a combination of methods for particle surface and size analysis[END_REF]. This very limited dissolution of Al NMs under acidic conditions therefore suggests that Al ions released from NMs do not contribute significantly to cytotoxic effects in our models.

AlCl 3

No genotoxic effects were observed in differentiated Caco-2 or HepaRG cells treated with AlCl 3 at concentrations up to 128 µg/mL Al content corresponding to 1.16 mg/mL AlCl 3 . At the concentrations of AlCl 3 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 [START_REF] Villarini | No evidence of DNA damage by co-exposure to extremely low frequency magnetic fields and aluminum on neuroblastoma cell lines[END_REF] who observed no genotoxicity in response to Al ions in neuroblastoma cells with the comet assay, as well as no cytotoxicity or oxidative stress. We previously showed that AlCl 3 induced other markers of oxidative stress but without observation of DNA damage (phosphoATM, phospho-p53 and H2AX) in HepaRG and HepG2 cells [START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF]. However, other studies have reported genotoxicity of AlCl 3 in human lymphocytes [START_REF] Lankoff | A comet assay study reveals that aluminium induces DNA damage and inhibits the repair of radiation-induced lesions in human peripheral blood lymphocytes[END_REF][START_REF] Paz | Evaluation of in vivo and in vitro toxicological and genotoxic potential of aluminum chloride[END_REF]. 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 AlCl 3 concentrations below 25 µg/mL, was associated with apoptosis [START_REF] Banasik | Aluminum-induced micronuclei and apoptosis in human peripheral-blood lymphocytes treated during different phases of the cell cycle[END_REF][START_REF] Lankoff | A comet assay study reveals that aluminium induces DNA damage and inhibits the repair of radiation-induced lesions in human peripheral blood lymphocytes[END_REF][START_REF] Lima | Genotoxic effects of aluminum chloride in cultured human lymphocytes treated in different phases of cell cycle[END_REF]. Apoptosis was also observed in hepatic cells but mostly in HepG2 compared to HepaRG cells [START_REF] Sieg | Aluminum in liver cells -the element species matters[END_REF]. Moreover it was shown that Al ions can induce oxidative DNA damage irrespective of the cell cycle phase [START_REF] Lankoff | A comet assay study reveals that aluminium induces DNA damage and inhibits the repair of radiation-induced lesions in human peripheral blood lymphocytes[END_REF]. 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 finger domains [START_REF] Hanas | Inhibition of transcription factor IIIA-DNA interactions by xenobiotic metal ions[END_REF][START_REF] Lankoff | A comet assay study reveals that aluminium induces DNA damage and inhibits the repair of radiation-induced lesions in human peripheral blood lymphocytes[END_REF]. Recently, a review on the genotoxicity of aluminum salts concluded that the absence of genotoxicity, even through oxidation [START_REF] Jenkinson | Critical Review of the Publications on the Genotoxicology of Aluminium Salts: 1990-2018[END_REF].

In our study, as the soluble fraction of AlCl 3 was always higher than that for Al 0 and Al 2 O 3 NMs, the effects observed for Al 0 and Al 2 O 3 NMs are not likely to be related to ion release in cell media. Although ECHA emphasized that the difference in the toxicological profile between soluble aluminum compounds and insoluble aluminum oxide may be explained by lower bioavailability of insoluble test compounds, it was recently shown that the content of Al in blood of rats treated orally was higher with Al 2 O 3 NMs than with AlCl 3 [START_REF] Krause | Aluminum and aluminum oxide nanomaterials uptake after oral exposure -a comparative study[END_REF]. 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.

ACCEPTED MANUSCRIPT / CLEAN COPY

Conclusions

In summary, despite the uptake and presence of Al NMs in the cytoplasm of differentiated Caco-2 and HepaRG cells, we have shown that Al 2 O 3 NMs did not induce any toxic effects in a panel of assays, with the exception of an increase of oxidative DNA damage in Caco-2 cells. Similarly, the assays performed with Al 0 NMs and AlCl 3 did not show a clear toxicity. However, it appears that Al 0 NMs can induce DNA damage when in contact with DNA as observed in the comet assay when testing interference. 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 cytotoxic effects resulting from the direct impact of the presence of intracellular particles. 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

ACCEPTED MANUSCRIPT / CLEAN COPY h at a concentration of 100 µg/mL. Three independent experiments were performed. Results are expressed as mean ± SD. 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. and ZnO NMs, and with the ionic salt control AlCl 3 . 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.

J o u r n a l P r e -p r o o f

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.

  vortexed right before measurement, to resuspend sedimented particles, and the DLS measurementswere analyzed by the Cumulant method. The Z-Ave and polydispersity index(PdI,) which represents the width of the distribution in terms of hydrodynamic diameter: PdI = (Standard deviation /mean)^2) are given in Table2. Measurements of the NM in the stock solution immediately following dispersion yielded Z-Ave = 254 ± 4 nm for Al 0 , 168 ± 3 nm for Al 2 O 3 and 233 ± 11 nm for ZnO. Although the three types of particles were supposed to have a similar primary size, we evidence that Al 2 O 3 NMs have a smaller Z-Ave than Al 0 and ZnO NMs. PdI was quite low (< 0.25) for all NMs in stock dispersion both at t=0 and after 24h. After 24h, the values for both Al-containing NMs were almost unchanged, indicating that no significant change of morphology of agglomeration state occurs within this period of time.However, the size of the ZnO NM appears about 20% lower after 24h, compared to t=0. This is also verified in the culture media, and could be due to partial solubilization of the ZnO. In constrast, the Z-Ave of the Al(0) NM is unchanged after 24H in all media. Al 2 O 3 NMs, exhibit a different behavior, with no significant change after 24h in stock solution, but a Journal Pre-proof slightly larger size in both cell media. This increase in size in not accompagnied with an increase in PdI, which could be due to protein adsorption.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[START_REF] Deloid | Preparation, characterization, and in vitro dosimetry of dispersed, engineered nanomaterials[END_REF] to evaluate the sedimentation of Al 0 and Al 2 O 3 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 fluid. The effective density ( eff ) of these agglomerates should first be measured to determine the colloidal behavior of such agglomerates. The effective densities determined for Al 0 NMs are 1.18 and 1.19 in DMEM and William's media respectively. Densities of 1.24 and 1.17 were obtained for Al 2 O 3 NMs in DMEM and William's cell culture media respectively. Applying the sedimentation model provided in DeLoid et al[START_REF] Deloid | Preparation, characterization, and in vitro dosimetry of dispersed, engineered nanomaterials[END_REF], we calculated the evolution of the concentration of NMs at the surface of cells with respect to time (Figure1). The sedimentation of Al 0 NMs was similar in both media, and after 24 h the concentration at the bottom of the well is approximately 650 µg/mL. The rate of sedimentation of Al 2 O 3 NMs was relatively slower, and the difference was significantly more pronounced in William's medium, with a deposited concentration of 450 µg/mL at the bottom of the well after 24 h.

Figure 1 :

 1 Figure 1: Time evolution over 24 h of the well-bottom concentration of (a) Al 0 NMs in DMEM; (b) Al 2 O 3 NMs in DMEM; (c) Al 0 NMs in William's medium; (d) Al 2 O 3 NMs in

  0 and Al 2 O 3 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 Al 0 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 Al 0 NMs were observed as isolated nanoparticle clusters in the cytoplasm proximal to the nucleus (Figure 2 and 3 C, full arrows). Observations of Al 0 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 Al 2 O 3 NMs was similar in both cell lines. While a perinuclear localization of Al 2 O 3 NMs was also seen, this occured less frequently than for Al 0 NMs (Figure 2 and 3, D and E). Even at the lowest concentration, Al 0 and Al 2 O 3 NMs were internalized through vesicle formation, most likely endocytosis, and accumulated in the cytoplasm of Caco-2 and HepaRG cells (Figure S 2).

Figure 2 :

 2 Figure 2: TEM images of differentiated Caco-2 cross-sections showing the uptake of Al 0 NMs (B, C) at 21 µg/cm 2 and Al 2 O 3 NMs (D,E) at 39 µg/cm 2 , after 24 h treatment compared to the negative control (A). Concentrations of 21 µg/cm 2 Al 0 NMs and 39 µg/cm 2 Al 2 O 3 NMs

Figure 3 :

 3 Figure 3: TEM images of differentiated HepaRG cross sections showing the uptake of Al 0 (B, C) at 21 µg/cm 2 and Al 2 O 3 (D,E) at 39 µg/cm 2 , after 24 h cell treatment compared to the negative control (A). Concentrations of 21 µg/cm 2 Al 0 NMs and 39 µg/cm 2 Al 2 O 3 NMs were

Figure 4 :

 4 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 Al 0 NMs, Al 2 O 3

Figure 5 :

 5 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 Al 0 NMs, Al 2 O 3 NMs and AlCl 3 as ionic salt control.

Figure 6 :

 6 Figure 6: Effects of Al-containing NMs on γH2AX level in Caco-2 and HepaRG Cells. A) Cells were treated for 24 h with Al 0 NMs, Al 2 O 3 NMs and AlCl 3 as ionic salt control, or

Figure 7 :

 7 Figure 7: Detection of DNA damage in differentiated Caco-2 and HepaRG cells treated 24 h

Figure 8 :

 8 Figure 8: Interference of Al 0 and Al 2 O 3 NMs with the alkaline and Fpg-modified comet assays. The interference of Al 0 and Al 2 O 3 NMs with DNA migration was assessed with

  modification of cell viability (RI value) was observed in either Caco-2 or HepaRG cells 0 , Al 2 O 3 , ZnO NMs and AlCl 3 . Compared to the negative control, no significant increase in the percentage of binucleated micronucleated (BNMN) cells was detected in either cell line. Similarly, no increase in micronucleated mononuleated cells or in polyploid cells was observed (data not shown).

  Ion release from NMs in cell culture media, or in intracellular compartments can contribute to cytotoxic effects in vitro. The soluble fraction of Al 0 and Al 2 O 3 NMs measured by ICP-MS demonstrated a very low solubility of Al 0 and Al 2 O 3 NMs in both cell media.

0and

  Al 2 O 3 NMs, and with AlC1 3 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 transformed foci per well (foci/well) were measured from 3 and 6 replicate cultures respectively, according to the OECD.

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  Table S 1B. Al content corresponding to the concentrations of Alcontaining NMs and AlCl 3 that were used are summarized in Table S

  0 and Al 2 O 3 NMs, suspensions were diluted in stock

	solution (ultra pure water + 0.05 % bovine serum albumin (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% HNO 3 , 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)

Table 1 :

 1 Summary of NM characteristics as reported by the supplier.

	NM	NM-code	Average particle size (nm)	a	SSA (m 2 /g) b	Purity	c	(g/cm 3 ) True density Bulk density	d	Morphology
	Al 0	NM-0015-HP	18		40-60	> 99%		0.008-0.20 2.70		spherical
		ACCEPTED MANUSCRIPT / CLEAN COPY

d Density was assessed by normal volumetric test

Table 2 :

 2 Physico-chemical characterization of Al 0 , Al 2 O 3 and ZnO NMs.

	Sample (100 µg/ml)	PdI	Z-Ave (d.nm)	PdI	Z-Ave (d.nm)
			Dispersion solution (0h)		Dispersion solution (24 h)
	Al	0	0.173 ± 0.004	254 ± 4	0.159 ± 0.026	253 ± 12
	Al 2 O 3	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)
	Al	0	0.176 ± 0.011	197 ± 2	0.156 ± 0.011	201 ± 1
	Al 2 O 3	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)
	Al	0	0.158 ± 0.008	240 ± 14	0.152 ± 0.007	246 ± 12
	Al 2 O 3	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

Table 3 :

 3 Ion release from Al 0 , Al 2 O 3 , ZnO NMs and AlCl 3 .

			NM	Dispersion
					DMEM + 10%	William's +
	NMs	concentration	stock solution
					FBS [%]	5% FBS [%]
			(µg/mL)	[%]
			25	1.30 ± 0.06	3.88 ± 0.13	2.42 ± 0.07
	Al	0	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
			25	0.24 ± 0.04	0.40 ± 0.03	0.32 ± 0.11
	Al 2 O 3	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
			25	89.60 ± 5.97	57.16 ± 5.97	23.35 ± 0.26
	AlCl 3	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
			25	29.21 ± 0.53	85.09 ± 5.30	54.99 ± 1.03
	ZnO	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

Table 4 :

 4 Detection of chromosomal damage in differentiated Caco-2 and HepaRG cells treated with Al-containing NMs. Cells were treated with increasing concentrations of Al

	0	,

Table 5 :

 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 Al

					Caco2		HepaRG
					%BNMN	RI (%)	% BNMN	RI (%)
	Control	0	5.6 ± 0.3	100 ± 0	3.6 ± 0.5	100 ± 0
				3	5.7 ± 0.4	97 ± 0.8	3.2 ± 0.3	97 ± 5.7
	Al	0	NMs	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
	[µg/cm 2 ]
				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
				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
	Al 2 O 3 NMs
				28	6.3 ± 1.4	96 ± 2.2	4.5 ± 0.8	109 ± 7.2
	[µg/cm 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
	AlCl 3	28	6.9 ± 0.5	103 ± 1.5	4.7 ± 1.5	114 ± 3.8
	[µg/ml]	40	6.9 ± 0.8	100 ± 1	4.3 ± 0.8	113 ± 3.3
	ZnO NMs	3	7 ± 0.8	100 ± 4.1	3.7 ± 0.4	96 ± 3.5
	[µg/cm 2 ]	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

Acknowledgements

The authors would like to thank Rachelle Lanceleur and Marie-Thérèse Lavault for technical assistance.

(MMS) was purchased by Acros Organics (Fairlawn, NJ). Dinophysistoxin-2 (DTX-2) was from the National Research Council Canada (NRCC, Ottawa, Canada). FBS) were supplied from Invitrogen Corporation (Illkirch, France). Dulbecco's modified Eagle's medium/Ham's F12 was from Invitrogen Corporation (Illkirch, France). Fetal bovine serum (FBS) was obtained by Dutscher, (Brumath France). Hydrocortisone hemisuccinate, Hyclone TM Dulbecco's Modified Eagle's Medium (DMEM)/high glucose and FBS for Caco-2 cells were purchased from Upjohn Pharmacia (Guyancourt, France), GE Healthcare Life Science (Logan, UT,

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).

Author 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 final manuscript.

Declaration of Competing Interest

The authors declare that they have no competing interests.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests

Highlights

 Al 0 and Al 2 O 3 NMs did not induce chromosomal damage in the micronucleus assay.

 Al 0 interference was observed in the comet assay.

 Al 2 O 3 induced DNA damage in the Fpg-modified comet assay although no significant ROS increase was observed.