Chiara Uboldi
email: chiara.uboldi@imbe.fr

Patricia Urbán
email: patricia.urban-lopez@ec.europa.eu

Douglas Gilliland
email: douglas.gilliland@ec.europa.eu

Edyta Bajak

Eugenia Vlsami-Jones

Jessica Ponti
email: jessica.ponti@ec.europa.eu

François Rossi
email: francois.rossi@ec.europa.eu

Eugenia Valsami-Jones
email: e.valsamijones@bham.ac.uk

Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts

Keywords:

Introduction

Titanium dioxide nanoparticles (TiO 2 NPs, titania) are currently used in a variety of applications ranging from the paper industry, where they confer whiteness and opacity in cellulose papers [START_REF] Huang | Fabrication and characterization of superhydrophobic high opacity paper with titanium dioxide nanoparticles[END_REF], to the production of antifogging and self-cleaning glasses and plastics [START_REF] Howarter | Self-cleaning and next generation anti-fog surfaces and coatings[END_REF]. TiO 2 NPs are also used as food color additives and flavor enhancers [START_REF] Weir | Titanium dioxide nanoparticles in food and personal care products[END_REF] and in various cosmetics owing to their ability to absorb and scatter UV light [START_REF] Wolf | Sunscreens-the ultimate cosmetic[END_REF][START_REF] Shi | Titanium dioxide nanoparticles: a review of current toxicological data[END_REF]. Due to their photocatalytical properties titania can additionally be employed in the degradation of pollutants in water treatment implants [START_REF] Chong | Recent developments in photocatalytic water treatment technology: a review[END_REF].

Nevertheless, the wide use of TiO 2 NPs, which naturally can occur in three different crystalline forms namely anatase, rutile and brookite, raised concern in the last years. Based on the evidence that titania can induce lung cancer in rats, the IARC (2010) classified TiO 2 NPs as possibly carcinogenic to humans (group 2B). In fact, lung tumors [START_REF] Xu | Involvement of macrophage inflammatory protein 1alpha (MIP1alpha) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying[END_REF], broncho-alveolar adenomas and cystic keratinizing squamous cell carcinomas [START_REF] Mohr | Pulmonary tumor types induced in Wistar rats of the so-called "19-dust study[END_REF] developed following inhalation and instillation of, respectively, rutile and anatase TiO 2 NPs.

In addition, nowadays several studies report that titania might be harmful to human health. Rutile titania impaired the viability of human amnion cells [START_REF] Saquib | Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in human amnion epithelial (WISH) cells[END_REF], skin fibroblasts and human epidermoid carcinoma cells [START_REF] Uchino | Effects of coating materials and size of titanium dioxide particles on their cytotoxicity and penetration into the cellular membrane[END_REF], but cytotoxicity was observed also in Chinese hamster fibroblasts [START_REF] Hamzeh | In vitro cytotoxicity and genotoxicity studies of titanium dioxide TiO2 nanoparticles in Chinese hamster lung fibroblast cells[END_REF]. Similarly, anatase TiO 2 NPs induced oxidative stress, apoptosis and impairment of the mitochondrial activity in in vitro humanderived glial and lung cells [START_REF] Aueviriyavit | Titanium dioxide nanoparticles-mediated in vitro cytotoxicity does not induce Hsp70 and Grp78 expression in human bronchial epithelial A549 cells[END_REF][START_REF] Huerta-García | Titanium dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in glial cells[END_REF]. Moreover, anatase exerted cytotoxic effects in Chinese hamster fibroblasts [START_REF] Chen | Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro[END_REF] and in murine osteocells [START_REF] Bernier | Preosteoblasts and fibroblasts respond differently to anatase titanium dioxide nanoparticles: a cytotoxicity and inflammation study[END_REF](Bernier et al., , 2010b)). However, anatase was not cytotoxic in human intestinal cells [START_REF] De Angelis | Comparative study of ZnO and TiO₂ nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells[END_REF], as well as in nasal mucosa cells [START_REF] Hackenberg | Intracellular distribution, geno-and cytotoxic effects of nanosized titanium dioxide particles in the anatase crystal phase on human nasal mucosa cells[END_REF] and in lymphocytes [START_REF] Hackenberg | Nanosized titanium dioxide particles do not induce DNA damage in human peripheral blood lymphocytes[END_REF].

Analogously, no conclusive results on the genotoxic potential exerted in vitro by anatase and rutile NPs have been reported. Increased DNA strand breaks and chromosome aberrations were detected in human intestinal, amniotic and epidermal cells [START_REF] Saquib | Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in human amnion epithelial (WISH) cells[END_REF][START_REF] Shukla | ROSmediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells[END_REF], and in Chinese hamster ovary and lung fibroblasts exposed to the two types of titania (Di Virgilio et al., 2010a, 2010b;[START_REF] Hamzeh | In vitro cytotoxicity and genotoxicity studies of titanium dioxide TiO2 nanoparticles in Chinese hamster lung fibroblast cells[END_REF]. Conversely, anatase TiO 2 NPs were not able to induce DNA damage in human nasal mucosa [START_REF] Hackenberg | Intracellular distribution, geno-and cytotoxic effects of nanosized titanium dioxide particles in the anatase crystal phase on human nasal mucosa cells[END_REF] and peripheral blood lymphocytes [START_REF] Hackenberg | Nanosized titanium dioxide particles do not induce DNA damage in human peripheral blood lymphocytes[END_REF].

Although many studies have been published showing experimental evidences of the toxic potential of TiO 2 NPs, the role played by the crystalline form of titania in the toxicity mechanisms is still unclear. In this study, we evaluated the effects induced in vitro by TiO 2 NPs in Balb/ 3T3 mouse fibroblasts which after exposure to carcinogens become tumorigenic forming morphologically transformed colonies (foci type III). Their ability to induce cancer in vivo is detectable by injecting the transformed cells in nude mice and observing their growth as sarcomas [START_REF] Kurzepa | Growth characteristics of tumors induced by transplantation into athymic mice of BALB/3T3 cells transformed in vitro by residue organics from drinking water[END_REF][START_REF] Saffiotti | Neoplastic transformation by quartz in the BALB/3T3/A31-1-1 cell line and the effects of associated minerals[END_REF]. This cell model was selected among Syrian Hamster Embryo (SHE) cells, C3H10T1/2 and Bhas42 in vitro models [START_REF] Kerckaert | Use of the Syrian hamster embryo cell transformation assay for determining the carcinogenic potential of heavy metal compounds[END_REF][START_REF] Isfort | Application of in vitro cell transformation assays to predict the carcinogenic potential of chemicals[END_REF][START_REF] Balls | A FRAME response to the draft report on alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010[END_REF] alternative to the use of animals able to detect morphological neoplastic transformation potential induced by chemicals and nanomaterials (NM).

In particular, we compared the effects exerted by two crystalline forms (anatase and rutile) of nanosized titania with their respective bulk materials, focusing not only on the cytotoxicity and the morphological transformation induced by TiO 2 NPs, but investigating also the genotoxicity of titania and their internalization in Balb/3T3 cells. The in vitro cytotoxicity was assayed performing the non-colorimetric colony forming efficiency (CFE) test, while cell transformation assay (CTA) evaluated morphological neoplastic transformation induced by anatase and rutile TiO 2 NPs. To investigate if TiO 2 NPs are able to induce chromosomal damage, we performed the cytokinesis-blocked micronucleus (CBMN) assay, and the uptake of titania in Balb/3T3 cells was investigated by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), which allowed us to perform a correlation between the amount (expressed as mass and number of particles) of TiO 2 NPs internalized and their toxic potential.

Materials and methods

Synthesis and characterization of TiO 2 NPs

Pure anatase and rutile TiO 2 NPs were obtained by hydrolysis and oxidation of TiCl 3 as previously described [START_REF] Cassaignon | From TiCl 3 to TiO 2 nanoparticles (anatase, brookite and rutile): thermohydrolysis and oxidation in aqueous medium[END_REF][START_REF] Valsami-Jones | Nanomaterial synthesis and characterization for toxicological studies: TiO 2 case study[END_REF][START_REF] Sweeney | Nano-titanium dioxide bioreactivity with human alveolar type-I-like epithelial cells: investigating crystalline phase as a critical determinant[END_REF].

Stock suspensions of nanosized anatase (An-10), nanosized rutile (Ru-10), bulk anatase (BAN) and bulk rutile (BRU) were characterized in deionized water by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). For TEM analysis, each sample was dropped onto ultrathin Formvar-coated 200-mesh copper grids (Ted Pella, Inc.; USA) and left to air dry before image acquisition was performed with a JEOL 2100 electron microscope (JEOL USA, Inc.; USA). Zeta-potential was also recorded for all the samples at pH 7. To further investigate their behavior, TiO 2 NPs were characterized by centrifugal liquid sedimentation (CLS) in deionized water and at increasing time points (0-24-48-72 h) incubation in complete cell culture medium. Before each analysis stock suspensions of TiO 2 NPs were bath-sonicated (10 min) and then aliquots were added to deionized water or to complete cell culture medium to obtain 0.1 mg/ml test suspensions. CLS characterization was performed by using the DC24000UHR centrifugal particle sedimentation (CPS Instruments; The Netherlands) operated at a speed of 30,000 ×g in an 8-24% wt sucrose density gradient.

Cellular system

Immortalized Balb/3T3 mouse fibroblasts (clone A31-1-1) purchased from Hatano Research Institute (Japan). Cells were cultivated under normal cell culture conditions (37 °C; 5% CO 2 ; 95% humidity) in complete cell culture medium composed of Modified Essential Medium (MEM) 1 × (Invitrogen; Italy), supplemented with 10% (v/v) fetal bovine serum -Australian origin (Invitrogen; Italy) and 1% (v/v) antibiotics (Invitrogen; Italy). Sub-confluent cells (70-80%) were passaged weekly in tissue culture-treated flasks (BD Falcon; Italy).

Colony forming efficiency (CFE) test

To investigate the potential in vitro cytotoxicity of TiO 2 NPs in Balb/ 3T3 mouse fibroblasts, colony forming efficiency (CFE) assay was performed as previously described [START_REF] Uboldi | Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts[END_REF]. Deionized water and sodium chromate (1000 μM) were used as solvent (C solv) and positive (C+) control, respectively. After TiO 2 NPs were bath-sonicated for 10 min, aliquots of the stock suspensions were directly pipetted into the culture dishes to obtain the desired test concentrations (1-5-10 μg/ml) and cells were exposed to titania for 24 h and 72 h.

The number of colonies was scored using the software-operated colony counter GelCount™ (Oxford Optronix Ltd.; UK). Data were normalized to the solvent control and expressed as % CFE [(average of treatment colonies / average of solvent control colonies) × 100]. The standard error of the mean (SEM) was calculated for 3 independent experiments and 3 replicates for each experimental point (SEM = standard deviation / square root of the number of replicates). Statistical significance was calculated by one-way ANOVA analysis with Bonferroni post hoc test (GraphPad Prism5 statistical software, GraphPad Inc.; USA).

Cytokinesis-blocked micronucleus (CBMN) test

To test the genotoxic potential of TiO 2 NPs, we performed the CBMN test as already described [START_REF] Uboldi | Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts[END_REF]. Cells were exposed to the fixed concentration of 10 μg/ml for each TiO 2 NPs. The data (3 independent experiments and 3 replicates for each experiment performed) were analyzed by manually scoring the presence of micronuclei (MN) in 1000 binucleated (BN) cells. The results are expressed as the number of binucleated micronucleated (BNMN) cells per 1000 binucleated cells (BNMN/1000 BN cells) ± SEM. Statistical analysis was performed by one-way ANOVA with Bonferroni post hoc test (GraphPad Prism5 statistical software, GraphPad Inc.; USA), and differences versus C solv (ultrapure water) were considered statistically significant at p b 0.05.

Cell transformation assay (CTA)

Cell transformation assay (CTA) was performed to test if TiO 2 NPs can induce morphological neoplastic transformation in Balb/3T3 fibroblasts since they become tumorigenic forming morphologically transformed colonies (foci type III). CTA is considered a powerful tool to investigate in vitro the morphological neoplastic transformation induced by chemicals and nanomaterials [START_REF] Vanparys | Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics[END_REF][START_REF] Ponti | Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential[END_REF]. Recently, its development into an OECD test guideline was recommended (EURL ECVAM Recommendation, 2012).

As previously described [START_REF] Uboldi | Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts[END_REF][START_REF] Ponti | Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential[END_REF], Balb/ 3T3 cells were seeded in 100 mm-Petri dishes (3.34 × 10 3 cells/ml; 6 ml/ dish; 3 independent experiments and 5 replicates per concentration) and exposed for 72 h to TiO 2 NPs (Table 1). Untreated Balb/3T3 cells and cells exposed to 4 μg/ml of 3-methylcholanthrene (Sigma-Aldrich; Italy) were used as negative and positive controls, respectively. After fixation (4% (v/v) of formaldehyde in PBS) and staining (10% (v/v) Giemsa solution in ultrapure water), the morphologically transformed colonies (type-III foci) were scored as described by the International Agency for Cancer research IARC Working-Group (IARC/NCI/EPA Working Group, 1985). Transformation results were expressed as transformation frequency (T f ) using the formula T f = [A / (B × C × D)], where A = total number of type III foci per treatment; B = CFE(%) / 100; C = Plating Efficiency (%) / 100; D = number of cell seeded multiplied per the number of plates; Plating Efficiency (%) = [(number of colonies formed in the control × 100) / 200], and 200 is the total number of cells initially seeded in each dish. Statistical analysis was performed applying the F-Fisher exact test and results were considered significant versus solvent control only when p b 0.05.

Uptake of TiO 2 NPs

To verify and quantify the uptake of TiO 2 NPs in Balb/3T3 mouse fibroblasts, the Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) technique was performed using a 7700 series ICP-MS (Agilent Technologies Inc.; Italy). 5 × 10 5 Balb/3T3 cells were seeded in 75 cm 2 tissue culture treated flasks (BD Falcon; Italy) in 10 ml of complete cell culture medium, and treated with a nominal concentration of 0.5 mg/ml TiO 2 NPs.

After 72 h exposure, the medium was collected for each test sample and stored at -20 °C. The cells were washed twice with 5 ml PBS, and each wash was collected in a separate tube and frozen for further analysis. Balb/3T3 cells were trypsinized, suspended with 9 ml of complete culture medium and then counted using a TC10 automated cell counter (Bio-Rad; Italy). After centrifugation, the supernatant was collected into a new tube and the dried pellet stored at -20 °C.

Before performing ICP-MS analysis, the frozen samples were restored at RT. To quantify the amount of Ti internalized, the cellular pellet was mineralized with Aqua Regia (HNO 3 :HCl, 1:3 v/v) and digested in a microwave (Discover SP-D; CEM SRL; Italy). Two independent experiments (3 replicates each experiment) were carried out and results were expressed as picograms (pg) of uptaken Ti/cell using the formula (A / B) × C, where A = [Ti], expressed as ppb, at the end of the experiment; B = total number of cells at the end of the experiment and C = conversion factor ppb to pg. We also calculated the number of internalized TiO 2 NPs/cell using the formula D/B, where D = number of TiO 2 NPs at the end of the experiment. D was calculated transforming the concentration of titania found in the cellular pellet in mass of nanoparticles. This value was transformed into number of nanoparticles knowing that titania are insoluble [START_REF] De Angelis | Comparative study of ZnO and TiO₂ nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells[END_REF] and assuming that most of the particles are spherical and well dispersed. Statistical analysis was performed applying t-test and results were considered significant when p b 0.05.

Results

3.1. Characterization of TiO 2 NPs: hydrodynamic diameter, PDI and Z-potential DLS measurements (Table 1) showed that the mean size diameter of as-synthesized An-10 and Ru-10 was 51.42 ± 0.35 nm and 134.40 ± 1.02 nm respectively while it was 333.80 ± 2.90 nm and 734.20 ± 15.56 nm for BAN and BRU respectively. Polydispersity index (PDI) for these samples were ranging between 0.17 ± 0.01 and 0.24 ± 0.04, indicating that TiO 2 NPs were relatively well dispersed in suspension. The Z-potential values at pH 7.0 for An-10 (-5.64 ± 0.77 mV), Ru-10 (+0.04 ± 0.13 mV) and BRU (-6.62 ± 3.90 mV) indicated a tendency to agglomerate/aggregate, whereas BAN resulted highly negative (-48.40 ± 1.04 mV) and stable.

TEM characterization (Fig. 1) indicated that An-10 is composed by primary particles of ovoid and spherical shape with a mean size diameter ranging from 11 nm to 18 nm, while Ru-10 showed more elongated particles ranging from 10 nm to 35 nm. BAN and BRU were highly aggregated and composed of particles with different geometry, whose size ranged from 60 nm to 400 nm (BAN) and from 250 nm to 600 nm (BRU).

Further physico-chemical characterization of TiO 2 NPs suspensions (100 μg/ml) in deionized water and in cell culture medium supplemented with 10% serum was performed by CLS (Table 2). Freshly prepared An-10 (t = 0 h) resulted in nanoparticles of the same mean size range either in water or in culture medium (49 ± 21 nm and 46 ± 15 nm, respectively), while a slight but not significant increase in its hydrodynamic diameter was observed after 24-48-72 h incubation. Similarly, Ru-10 showed no significant increase in mean diameter when the value in water was compared to the particle size measured in the presence of serum proteins and at increasing time points. Analogously, bulk particles did not significantly increase their mean size in the presence of serum proteins (BAN = 260 ± 20 nm and BRU = 755 ± 180 nm) compared to deionized water (243 ± 16 nm and 717 ± 55 nm, respectively).

In deionized water, DLS measurements of An-10, RU-10 and BRU are in agreement with CLS data except for the hydrodynamic diameter of BAN. When TiO 2 NPs were diluted in complete culture medium, both Ru-10 and BAN displayed a significantly different hydrodynamic diameter (p b 0.01 and p b 0.001, respectively) in comparison to the native stock suspensions. Moreover, CLS analysis showed that at increasing time points the presence of serum did not significantly change the mean size diameter of TiO 2 NPs.

In vitro cytotoxicity of TiO 2 NPs: evaluation of the clonogenic potential

At 24 h exposure, none of the investigated TiO 2 NPs significantly impaired the formation of Balb/3T3 colonies. Compared to deionized water, which was used as C solv (100% CFE ± 3.02), a slight but not significant CFE reduction (Fig. 2) was observed in Balb/3T3 cells exposed to 5 μg/ml Ru-10 (95.96% CFE ± 5.99) and to 5 μg/ml and 10 μg/ml BRU (97.17% CFE ± 5.21 and 96.97% CFE ± 2.6, respectively).

In contrast, 72 h exposure of Balb/3T3 cells to Ru-10 and BRU at 5 μg/ml and 10 μg/ml doses significantly reduced the CFE: 5 μg/ml Ru-10 impaired the CFE to 73.7% CFE ± 3.62 and 10 μg/ml Ru-10 to 80.22% CFE ± 4.81. Similarly, 5 μg/ml and 10 μg/ml BRU affected the formation of Balb/3T3 colonies to 78.24% CFE ± 2.78 and to 79.38% CFE ± 3.97, respectively.

A non-significant impairment of CFE was observed following 72 h exposure to An-10. In fact, compared to the C solv (100% CFE ± 5.84), 5 μg/ml and 10 μg/ml reduced the scored Balb/3T3 colonies to 92.85% CFE ± 3.38 and to 89.64% CFE ± 7.0, respectively. In addition, a statistically non-significant cytotoxic effect was also observed in the presence of 1 μg/ml Ru-10 (82.21% CFE ± 8.54) and 1 μg/ml BRU (92.0% CFE ± 7.53). Taken together, our results indicated that the crystalline form (rutile) might be responsible for TiO 2 NPs cytotoxicity in Balb/3T3 mouse fibroblasts.

Genotoxicity and morphological neoplastic transformation induced by TiO 2 NPs

Since the concentration of 10 μg/ml resulted sub-toxic or non-toxic for all the tested titania, it was selected as the most suitable concentration for genotoxicity and morphological neoplastic transformation tests. Using CBMN test, we observed that in the presence of TiO 2 NPs, the nuclear division index (NDI; Fig. 3A) did not significantly change as compared to C solv (NDI = 2.02) and confirmed that, for each experimental condition, Balb/3T3 cells replicated at least one time during the exposure period, as recommended by the Test Guideline 487 (OECD, 2010). The analysis of micronuclei (MN) induction, as indicator of chromosomal damage, showed that a statistical significant (p b 0.001) result in comparison with C solv was observed only after exposure to 10 μg/ml Ru-10 and to the positive control 0.15 μg/ml MMC (Fig. 3B).

We further investigated the morphological neoplastic transformation potential of TiO 2 NPs by performing the in vitro cell transformation assay (Fig. 4). Compared to C solv (Tf = 1.4 × 10 -4 ± 0.1) Ru-10 induced statistically significant dose-dependent increasing transformation frequency. Similarly, BRU resulted transforming Balb/3T3 cells, and the highest Tf was scored at the highest tested dose (3.8 × 10 -4 ± 0.2 at 10 μg/ml). Although An-10 and BAN slightly enhanced type-III foci formation, their effect was not statistically significant.

3.4. TiO 2 NPs-Balb/3T3 interaction: uptake quantification Balb/3T3 cells were exposed to treatment suspensions at the theoretical concentration of 0.5 mg/ml for 72 h and in order to quantify the real titanium concentration, the treatment suspensions were analyzed by ICP-MS. As shown in Fig. 5A, Ru-10 (real concentration 0.51 ± 0.01 mg/ml) seems to be the most consistent with the expected theoretical concentration; the measured An-10 and BAN (0.55 ± 0.01 mg/ml and 0.44 ± 0.01 mg/ml, respectively) were slightly different from the theoretical concentration, but the difference was not statistically significant. BRU, in contrast, appeared as the treatment suspension that more significantly (p b 0.001) differed from the theoretical concentration (0.71 ± 0.02 mg/ml versus 0.5 mg/ml expected). This behavior was probably due to the higher size distribution and higher tendency to aggregate as compared to the other titania tested (Tables 1,2).

Interestingly, when the number of TiO 2 NPs uptaken per cell was calculated (Fig. 5B), nanosized anatase and rutile titania were internalized more significantly (p b 0.001) compared to their bulk counterparts: An-10 resulted being the most uptaken nanoparticles (1.07 × 10 8 ± 2.56 × 10 6 NPs), followed by Ru-10 (1.93 × 10 7 ± 1.44 × 10 6 NPs), BAN (3.26 × 10 5 ± 6.6 × 10 3 NPs) and BRU (7.14 × 10 4 ± 5.3 × 10 3 NPs). Additionally, the number of anatase TiO 2 NPs, either in the nanorange or in the bulk form, was statistically significant (p b 0.001) compared to rutile. Coherently, the quantification of the amount of Ti, expressed as pg/ml, still present in the collected supernatant at the end of the exposure period (Fig. 5C) showed that BAN and BRU contained more Ti (5246.17 ± 114.03 pg/ml

Table 2

CLS disc centrifuge characterization of TiO 2 NPs. The mean size diameter was determined in deionized water (A) and in complete cell culture medium (B) at the concentration of 100 μg TiO 2 NPs/ml; nominal density for An-10 is 3.89 g/cm 3 and for Ru-10 is 4.23 g/cm 3 . and 5895.15 ± 110 pg/ml, respectively) as compared to An-10 (299.68 ± 27 pg/ml) and Ru-10 (1291.48 ± 63 pg/ml). Consistently, the amount of Ti (pg/cell) internalized by Balb/3T3 mouse fibroblasts (Fig. 5D) was higher in Ru-10 and An-10 (5.06 ± 0.1 pg/cell and 4.15 ± 0.1 pg/cell, respectively) than in BAN (0.69 ± 0.1 pg/cell) and BRU (0.52 ± 0.06 pg/cell). Taken together, the uptake quantification data showed a correlation with the hydrodynamic diameter of the particles with a size-dependent pattern. The nanosized titania, which displayed the lower hydrodynamic diameter as shown in Table 2, were more easily internalized by Balb/ 3T3 than their respective bulks, indicating that size plays a role in the process. Interestingly, although An-10 was uptaken by Balb/3T3 mouse fibroblasts as efficiently as Ru-10, the internalization of the particles did not correlate with the toxicity data, which showed indeed a slightly more significant effect in the presence of nano-rutile titania, thus suggesting that the toxicity is triggered by the crystalline form of TiO 2 NPs.

A

Discussion

Although TiO 2 NPs are currently used in numerous industrial applications, a careful evaluation of their toxic potential is still needed. In this context, we studied the potential cytogenotoxicity, morphological neoplastic transformation and internalization of TiO 2 NPs, and we showed that their crystalline form is a key parameter in their toxicity effect in Balb/3T3 fibroblasts. By colony forming efficiency assay, under our experimental conditions, cytotoxicity was not observed at 24 h exposure in the presence of titania, whereas at 72 h incubation 5 and 10 μg/ml nanosized and bulk rutile titania significantly affected the in vitro formation of Balb/3T3 colonies. Similar effects are already reported in the literature (Table 3). [START_REF] Sweeney | Nano-titanium dioxide bioreactivity with human alveolar type-I-like epithelial cells: investigating crystalline phase as a critical determinant[END_REF] reported that the membrane integrity of TT1 human alveolar type-I-like epithelial cells was not affected by nanosized anatase and rutile titania, the same that was used in the present study. In contrast, Ru-10 seemed to have a greater pro-inflammatory activity because it stimulated a higher IL-6 and MCP-1 secretion compared to An-10 (Sweeney et al., 2014). At 1-5-10 μg/ml by MTT assay, [START_REF] Saquib | Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in human amnion epithelial (WISH) cells[END_REF] demonstrated that rutile TiO 2 NPs impaired the viability of WISH human amnion cells, and the toxicity at 24 h was comparable to our CFE results at 72 h. These different results can be explained taking into consideration the size of the particles: while [START_REF] Saquib | Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in human amnion epithelial (WISH) cells[END_REF] studied TiO 2 NPs with hydrodynamic diameter of 30 nm, our rutile titania ranged from 60 to 80 nm (Ru-10) to 730-750 nm (BRU) and that could have deferred the cytotoxic effect.

Our results show that the cytotoxicity of TiO 2 NPs might not be simply triggered by the size of the particles, but that the crystalline form plays a role as well. In fact, while we measured a significantly higher internalization of anatase TiO 2 NPs, it was the rutile crystalline form that induced cytotoxicity, genotoxicity and morphological transformation in Balb/3T3 fibroblasts. This effect was already reported in V79 cells, where within 24 h rutile TiO 2 nanorods (420 nm) reduced the cell survival more severely than 365-460 nm anatase ones [START_REF] Hamzeh | In vitro cytotoxicity and genotoxicity studies of titanium dioxide TiO2 nanoparticles in Chinese hamster lung fibroblast cells[END_REF]. Nevertheless, size-related cytotoxic effects exerted by rutile TiO 2 NPs in normal human skin fibroblasts, in Chinese hamster ovary and in rat basophilic leukemia cells were also reported. As shown by [START_REF] Uchino | Effects of coating materials and size of titanium dioxide particles on their cytotoxicity and penetration into the cellular membrane[END_REF], 20 nm rutile titania resulted more cytotoxic than 250 nm ones, and the effect positively correlated with the amount of particles internalized. Similarly, by ICP-MS we also detected a more significant internalization of nano-rutile TiO 2 NPs compared to its bulk, which could explain the higher toxicity induced by Ru-10 compared to BRU.

Under our experimental conditions, we showed that Balb/3T3 colony formation is not affected upon exposure to low concentrations (1-5-10 μg/ml) of bulk and nano-anatase titania. Similarly, [START_REF] Kim | Comparative cytotoxicity of Al 2 O 3 , CeO 2 , TiO 2 and ZnO nanoparticles to human lung cells[END_REF] reported that, up to 500 μg/ml, anatase TiO 2 NPs were not able to inhibit A549 and L-132 colony formation. Moreover, 15 nm TiO 2 NPs (10-100 μg/ml) increased the survival of NIH-3T3 cells as shown by CFE assay [START_REF] Huang | Disturbed mitotic progression and genome segregation are involved in cell transformation mediated by nano-TiO 2 long-term exposure[END_REF]. Taken together these results confirm that the crystalline structure has a key role in the cytotoxicity of TiO 2 NPs.

In contrast, previous studies reported that also anatase titania exert cytotoxic effects (Table 3). Cytotoxicity was detected in A549 cells exposed to anatase TiO 2 NPs; analogously, anatase titania affected the mitochondrial integrity of human-derived glial cells [START_REF] Huerta-García | Titanium dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in glial cells[END_REF] and V79 hamster lung fibroblasts [START_REF] Chen | Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro[END_REF].

In addition to their cytotoxic potential, TiO 2 NPs were already reported to induce genotoxic effects (Table 3). Anatase TiO 2 NPs of 20 nm slightly enhanced MN formation in binucleated CHO-K1 cells after 24 h treatment (Di Virgilio et al., 2010aVirgilio et al., , 2010b)), and in HaCaT cells 10 μg/ml of 200 nm TiO 2 NPs significantly increased the MN rate [START_REF] Jaeger | Oxidative stress-induced cytotoxic and genotoxic effects of nano-sized titanium dioxide particles in human HaCaT keratinocytes[END_REF]. Additionally, Shukla et al. reported that the dose- Based on the real concentrations measured, the number of TiO 2 NPs uptaken in each cell was calculated (B) and showed that An-10 and Ru-10 were internalized more efficiently (***p b 0.001) than BAN and BRU; moreover, the number of An-10 TiO 2 NPs uptake per Balb/3T3 cell was significantly higher than Ru 10 ( ### p b 0.001) and BAN particles were taken up more ( § § § p b 0.001) than BRU. Accordingly, the ICP-MS quantification of the amount of Ti, expressed as pg/ml, in the collected supernatant (C) revealed that bulk materials and the rutiles were the less internalized titania (***p b 0.001). The quantification of the amount of Ti (pg/cell) uptaken by the total amount of Balb/3T3 mouse fibroblasts (D) after 72 h exposure showed that nanosized TiO 2 NPs were internalized more (***p b 0.001) than bulk, but interestingly showed that Ti from Ru-10 was significantly higher ( # p b 0.05) than An-10. Mean of 2 independent experiments ± SEM.

dependent increase in MN frequency and the induction of oxidative DNA lesions observed in A431 human epidermal cells were correlated to an enhanced lipid peroxidation following exposure to 170 nm anatase TiO 2 NPs [START_REF] Shukla | ROSmediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells[END_REF]. Moreover, Falck and co-authors (2009) showed that anatase TiO 2 NPs (hydrodynamic diameter below 20 nm) enhanced the MN frequency in human derived bronchial cells BEAS-2B.

Our results partially confirmed these data, as we observed MN induction in Balb/3T3 at longer exposure periods (48 h), and only the chromosome aberrations induced by Ru-10 were statistically significant. In contrast, nano-rutile TiO 2 NP filaments, with primary size 10 × 40 nm, were reported to not increase MN frequency in BEAS-2B cells [START_REF] Falck | Genotoxic effects of nanosized and fine TiO 2[END_REF].

Additionally, we showed that the morphological neoplastic transformation is more related to the crystalline form than to the size of the particles: rutile TiO 2 NPs were able to significantly induce type-III foci formation in Balb/3T3 cells, whereas anatase TiO 2 NPs did not. To our knowledge this is the first in vitro study directly highlighting the neoplastic transformation potential of TiO 2 NPs, as previous investigations reported the same effect but via DNA damage analysis and, therefore, via a secondary genotoxic mechanism.

Additionally, we demonstrated that rutile and anatase TiO 2 NPs are internalized by Balb/3T3 cells, showing thus that the reduced colony forming efficiency and the formation of MN and type-III foci were due to a direct interaction between titania and cells. However, while we observed that the cytotoxic and genotoxic potential of TiO 2 NPs can be associated with their crystalline form, the uptake of TiO 2 NPs seemed to depend on their size. The nanosized An-10 and Ru-10, which by DLS were shown to have a smaller hydrodynamic diameter compared to their bulk counterparts, were significantly more uptaken compared to BAN and BRU. Even if further studies on NPs dosimetry are necessary to better understand kinetics of nanomaterials and to measure the real administrated, delivered and internalized concentrations [START_REF] Cohen | Interactions of engineered nanomaterials in physiological media and implications for in vitro dosimetry[END_REF][START_REF] Deloid | Estimating the effective density of engineered nanomaterials for in vitro dosimetry[END_REF], our ICP-MS data are in good agreement with previous published results. Aggregates/agglomerates of rutile TiO 2 NPs were observed by TEM in cytoplasmic vesicles in A549 [START_REF] Aueviriyavit | Titanium dioxide nanoparticles-mediated in vitro cytotoxicity does not induce Hsp70 and Grp78 expression in human bronchial epithelial A549 cells[END_REF] and WISH cells [START_REF] Saquib | Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in human amnion epithelial (WISH) cells[END_REF]. [START_REF] Shukla | ROSmediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells[END_REF] and [START_REF] Hackenberg | Intracellular distribution, geno-and cytotoxic effects of nanosized titanium dioxide particles in the anatase crystal phase on human nasal mucosa cells[END_REF] detected TiO 2 NPs in the nucleus of, respectively, human epidermal cells and human nasal mucosa cells, while in BEAS-2B cells the massive presence of particles in the perinuclear region was associated to an altered gene transcription induced by titania that, consequently, blocked the nuclear pores [START_REF] Park | Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells[END_REF]. Taken together, our results showed that the crystalline form has a role in toxicity in Balb/3T3 mouse fibroblasts. Rutile titania was slightly more toxic than anatase TiO 2 NPs. These results might be explained taking into account the different reactivity of the two crystalline forms we have investigated. In fact, rutile TiO 2 NPs are more photocatalytic than anatase and consequently, are able to generate higher amounts of oxygenated free radicals on their own surface [START_REF] Fenoglio | Non-UV-induced radical reactions at the surface of TiO 2 nanoparticles that may trigger toxic responses[END_REF][START_REF] Lipovsky | The different behavior of rutile and anatase nanoparticles in forming oxy radicals upon illumination with visible light: an EPR study[END_REF]. Moreover, since the ability of several types of nanoparticles to trigger genotoxic effects has been demonstrated to be linked to their ROS induction [START_REF] Magdolenova | Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles[END_REF], this can explain the results we have obtained by exposing Balb/3T3 mouse fibroblasts to TiO 2 NPs. Nevertheless, other physicochemical properties (such as size, mono/polydispersity or surface charge) might play a synergistic role in the differences existing between the mechanism of toxicity of rutile and anatase.

Results available in the literature are difficult to compare mainly due to different factors that can alter the properties of nanomaterials such as their source, the synthesis protocol used, size, coating of NPs and their behavior in culture media. Therefore, further investigation on cytogenotoxicity of titania using a methodological approach linked to a systematic physico-chemical characterization is still required.

Transparency document

The Transparency Document associated with this article can be found, in online version.

Fig. 1 .

 1 Fig. 1. TiO 2 NPs stock suspensions shape and size determination. TEM micrographs of the as-synthesized TiO 2 NPs indicated that An-10 (A) is composed by egg-shaped particles of 11-18 nm, while Ru-10 (B) is more elongated and the particles have a mean size diameter of 10-35 nm. BAN (C) and BRU (D) were highly aggregated and composed of particles with different geometries.

Fig. 2 .

 2 Fig. 2. Cytotoxicity induced by TiO 2 NPs in Balb/3T3 mouse fibroblasts. Colony forming efficiency (CFE) was performed on Balb/3T3 cells exposed for 24 h and 72 h to increasing concentrations (1-5-10 μg/ml) of TiO 2 NPs. Cytotoxicity was not observed after 24 h exposure, while at 72 h 5 μg/ml and 10 μg/ml Ru-10 and BRU significantly impaired the formation of Balb/3T3 colonies. An-10 and BAN did not exert any cytotoxic effect even after 72 h incubation. The positive control sodium chromate (1000 μM) induced complete cell death (0% CFE). Mean of 3 independent experiments including 3 replicates each ±SEM. Data were compared to C solv (deionized water, 100% CFE) and evaluated by one-way ANOVA with Bonferroni post hoc analysis. **p b 0.01. Fig. 3. Genotoxic potential of TiO 2 NPs by CBMN assay. The Nuclear Division Index (A) demonstrated that Balb/3T3 have divided at least one time during exposure to TiO 2 NPs (24 h exposure with 10 μg/ml titania). The number of binucleated micronucleated cells (BNMN) scored in 1000 binucleated cells containing one of more micronuclei (B) indicated that An-10, Ru-10 and BRU induced MN formation, but only Ru-10 was statistically significant. The statistical significance was evaluated by one-way ANOVA with Bonferroni post hoc analysis in respect to C solv. Mean of 3 independent experiments and 3 replicates each ± SEM. Mitomycin C (0.1 μg/ml) was used as positive control (C+). ***p b 0.001.

Fig. 5 .

 5 Fig. 5. TiO 2 NPs-cell interaction: quantification by ICP-MS. Compared to the theoretical (0.5 mg/ml), the measured concentration of TiO 2 NPs in the treatment suspensions (A) was significantly different only in BRU (***p b 0.001).Based on the real concentrations measured, the number of TiO 2 NPs uptaken in each cell was calculated (B) and showed that An-10 and Ru-10 were internalized more efficiently (***p b 0.001) than BAN and BRU; moreover, the number of An-10 TiO 2 NPs uptake per Balb/3T3 cell was significantly higher than Ru 10 ( ### p b 0.001) and BAN particles were taken up more ( § § § p b 0.001) than BRU. Accordingly, the ICP-MS quantification of the amount of Ti, expressed as pg/ml, in the collected supernatant (C) revealed that bulk materials and the rutiles were the less internalized titania (***p b 0.001). The quantification of the amount of Ti (pg/cell) uptaken by the total amount of Balb/3T3 mouse fibroblasts (D) after 72 h exposure showed that nanosized TiO 2 NPs were internalized more (***p b 0.001) than bulk, but interestingly showed that Ti from Ru-10 was significantly higher ( # p b 0.05) than An-10. Mean of 2 independent experiments ± SEM.

Table 1

 1 Characterization of TiO 2 NPs by Dynamic Light Scattering. The hydrodynamic diameter and the Z-potential at pH 7.0 were measured for TiO 2 NPs stock suspensions diluted in deionized water.

	Sample name	Hydrodynamic diameter (nm)	PDI a	Z-pot (mV) at pH 7.0
	An-10	51.42 ± 0.35	0.24 ± 0.04	-5.64 ± 0.77
	BAN	333.80 ± 2.90	0.19 ± 0.02	-48.40 ± 1.04
	Ru-10	134.40 ± 1.02	0.17 ± 0.01	+0.04 ± 0.13
	BRU	734.20 ± 15.56	0.24 ± 0.03	-6.62 ± 3.90

a Polydispersity index.

Table 3

 3 Toxicological effects of anatase and rutile titania in different sizes as reported in the literature.

	Cell line	Crystalline structure and size	Effects observed	Reference
	RBL-2H3 Rat basophilic leukemia cells	Rutile 20 nm and 250 nm	Size-related cytotoxicity	Uchino et al., 2011
	CHO Chinese hamster ovary cells	Rutile 20 nm and 250 nm	Size-related cytotoxicity	Uchino et al., 2011
	V79	Anatase 360-460 nm Rutile 420 nm	Rutile induced more cytotoxicity (decreased cell viability at concentrations ≥10 μg/ml) and apoptosis than anatase; anatase 460 nm was the most genotoxic	Hamzeh and Sunahara, 2013
	Chinese hamster lung fibroblasts		Time-and concentration-dependent impairment of cell
		Anatase 75 nm	viability; genotoxicity was detected at concentrations	Chen et al., 2014
			≥100 μg/ml
	RAW 264.7 Mouse macrophage cell line	Rutile 30-40 nm	Dose-dependent cytotoxicity and pro-inflammatory activity	Palomäki et al., 2010
	bmDC Murine bone marrow-derived dendritic cells	Rutile 30-40 nm	Dose-dependent cytotoxicity and pro-inflammatory activity	Palomäki et al., 2010
	MC-3T3 Murine pre-osteoblasts	Anatase 200-400 nm	Dose-related cytotoxicity; enhanced IL-6 secretion at concentrations ≥20 μg/ml	Bernier et al., 2012
	L929 Mouse fibroblasts	Anatase 200-400 nm	Dose-related cytotoxicity; enhanced IL-6 secretion at concentrations ≥500 μg/ml	Bernier et al., 2012
	UMR 106 Rat osteosarcoma-derived cells	Anatase 21 nm	Dose-dependent cytotoxicity detected at concentrations ≥25 μg/ml	Di Virgilio et al., 2010a, 2010b
	CHO-K1 Chinese hamster ovary cells	Anatase 21 nm	Dose-dependent cytotoxicity detected at concentrations ≥5 μg/ml; slight MN formation	Di Virgilio et al., 2010b
	NIH3T3 Mouse embryonic fibroblasts	Anatase 15 nm	No cytotoxicity observed	Huang et al., 2009
	A549 Human alveolar carcinoma cells	Anatase 20-40 nm Anatase 499-2865 nm Rutile 321-4235 nm	Low impairment of colony formation up to 500 μg/ml Dose-related cytotoxicity and mitochondrial impairment	Kim et al., 2010 Aueviriyavit et al., 2012
	WISH Human amnion epithelial cells	Rutile 30.6 nm (TEM), 13-308 nm (DLS)	Dose-dependent impairment in the cell survival	Saquib et al., 2012
	HaCaT Human keratinocytes	Anatase 200 nm	Significant MN formation after 24-48 h and oxidative stress after 4 h exposure	Jaeger et al., 2012
	NHSF Normal human skin fibroblasts	Rutile 20 nm and 250 nm	Size-related cytotoxicity	Uchino et al., 2011
	U373 Human glioblastoma astrocytoma cells	Anatase 420 nm	Induction of oxidative stress and mitochondrial depolarization	Huerta-García et al., 2014
	Caco-2
	Human epithelial colorectal adenocarcinoma	Anatase 200 nm	No cytotoxic effects	De Angelis et al., 2013
	cells
	TT1 Human alveolar type-I-like epithelial cells	Anatase 10-11 nm Rutile 9-12 nm	No cytotoxicity up to 100 μg/ml; dose-related IL-6 release, slightly greater following exposure to rutile	Sweeney et al., 2014
	Primary human nasal epithelial cells	Anatase 15-30 nm	No cytotoxicity nor genotoxicity detected	Hackenberg et al., 2010
	Freshly isolated human peripheral blood lymphocytes	Anatase 15-30 nm	No significant reduction of cell viability; no evidence for genotoxicity up to 200 μg/ml	Hackenberg et al., 2011
	A431 Human epidermal cells	Anatase 125 nm	Concentration and time dependent impairment of cell formation viability; dose-related DNA damage; significant MN	Shukla et al., 2011
	BEAS-2B Human bronchial epithelial cells	Anatase b25 nm Rutile fibers 10 × 40 nm	Anatase reduced cell viability more severely than rutile; dose-related MN formation after exposure to rutile	Falck et al., 2009
	L132 Normal human lung cells	Anatase 20-40 nm	No impairment of cell viability and of colony formation	Kim et al., 2010

C.Uboldi et al. / Toxicology in Vitro 31 (2016) 137-145

Acknowledgments

The authors would like to acknowledge Dr. Deborah Berhanu for the TiO 2 NPs synthesis and Dr. Fabio Franchini for performing the ICP-MS analysis.

Funding

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 214478 (NanoReTox).


Dupuy

N'kaoua Bernard

Sauzeon

Ph.D Lucile Dupuy
email: lucile.dupuy@u-bordeaux.fr

Role of cognitive resources on everyday functioning among oldest old physically frail

Keywords: aging; physical functioning, cognitive resources, daily life activities, compensation

Background: Everyday functioning becomes a challenge with aging, particularly among frail oldest-old adults. Several factors have been identified as influencing everyday activities realization, including physical and cognitive functioning. However, the influence of cognitive resources as a compensatory factor in the context of physical frailty deserves further consideration.

Aims: This study aims to investigate in older adults physically frail the possible compensatory role of cognitive resources to perform everyday tasks.

Methods: Two groups of community-dwelling old participants (n=26 per group) matched for their age and cognitive resources, have been drawn according to their level of physical functioning. Two measures of everyday functioning have been assessed: one self-reported by the participant (the IADL scale) and one performance-based measure (the TIADL tasks).

Results: Participants performed equally the TIADL tasks irrespective of their physical condition. Contrariwise, participants with low physical functioning reported more everyday difficulties than their counterparts with a high level of physical functioning. Additionally, regressions analyses revealed differential influence of cognitive resources on performance and reported measures of everyday functioning.

Discussion: Our data suggests that cognitive resources are more strongly involved in the performance-based IADL measure in situation of physical frailty. Additionally, for participants with low physical functioning, lower cognitive resources are associated with more perceived difficulties in everyday life.

Conclusion:

These results highlight the compensatory role of cognitive resources in physically frail older adults, and suggest that an overestimation of everyday difficulties compared to performance on IADL tasks is an early indicator of physical decline and cognitive compensation.

BACKGROUND

Aging is a multifactorial process, which is influenced by a large number of physical, psychological and social variables. While many persons experience healthy aging without significant impairments, sensory, motor and cognitive decline can occur with age. In any case, the capacity to perform activities of daily living can be affected. In this study, we focus on the compensatory role of cognitive resources on independent everyday functioning among older adults who have reduced physical functioning.

Independent everyday functioning and its assessment

Independent everyday functioning, commonly called functional status, refers to the individual's abilities to autonomously perform activities of daily life (ADL) [START_REF] Diehl | Everyday Competence in Later Life: Current Status and Future Directions[END_REF][START_REF] Heyl | Managing daily life with age-related sensory loss: Cognitive resources gain in importance[END_REF]. ADLs include basic (BADLs) and instrumental (IADLs) activities of daily living [START_REF] Lawton | Assessment of Older People: Self-Maintaining and Instrumental Activities of Daily Living[END_REF][START_REF] Gold | An examination of instrumental activities of daily living assessment in older adults and mild cognitive impairment[END_REF]. The former refer to basic, physical, self-care tasks, such as ambulating, dressing, grooming, toileting, eating, etc. The latter activities are more complex self-care tasks, such as meal preparation, medication and financial management, etc. Hence, IADLs entail more cognitively complex tasks than BADLs [START_REF] Lawton | Assessment of Older People: Self-Maintaining and Instrumental Activities of Daily Living[END_REF]. The IADL-related abilities are often assessed through self-reports or proxy ratings of an individual's ability to perform activities. IADL questionnaires commonly used in the older adult population include the Multilevel Assessment Instrument [START_REF] Lawton | A Research and Service Oriented Multilevel Assessment Instrument[END_REF], the SF-36 [START_REF] Ware | The MOS 36-Item Short-Form Health Survey (SF-36): I. Conceptual Framework and Item Selection[END_REF] and the OARS Multidimensional Functional Assessment Questionnaire [START_REF] Fillenbaum | Multidimensional Functional Assessment of Older Adults : The Duke Older Americans Resources and Services Procedures[END_REF]. As these questionnaires can generate biases and inaccuracies in the informant's perceptions, they are increasingly being complemented with objective performance measures of physical and cognitive tasks important for everyday functioning, such as the Timed Instrumental Activities of Daily Living test (TIADL [START_REF] Owsley | Timed Instrumental Activities of Daily Living Tasks: Relationship to Cognitive Function and Everyday Performance Assessments in Older Adults[END_REF]; for a review of such ADL measures, see [START_REF] Moore | A review of performance-based measures of functional living skills[END_REF]). To be noted, while with cognitively impaired older adults, a discrepancy has been observed between self-reported and objective measure of everyday functioning [START_REF] Suchy | Instrumental activities of daily living among community-dwelling older adults: Discrepancies between self-report and performance are mediated by cognitive reserve[END_REF], in cognitively healthy old people, self-reported ADL measures still remain strongly related to objective measures of everyday functioning [START_REF] Burton | Cognitive Functioning and Everyday Problem Solving in Older Adults[END_REF][START_REF] Bravell | Self-reported activities of daily living and performance-based functional ability: a study of congruence among the oldest old[END_REF].

Main underpinnings of age-related changes in independent everyday functioning

Autonomy in daily living has been shown to be influenced by a number of factors. First, ADL performance is related to socio-demographic variables, such as age, gender, education, and marital status [START_REF] Diehl | Everyday Competence in Later Life: Current Status and Future Directions[END_REF][START_REF] Pruchno | A Longitudinal Examination of the Effects of Early Influences and Midlife Characteristics on Successful Aging[END_REF][START_REF] Mejía | Successful Aging as the Intersection of Individual Resources, Age, Environment, and Experiences of Well-being in Daily Activities[END_REF]. For instance, over a longitudinal study with more than 3,000 participants, authors found that women with an advanced age and lower education were more likely to develop poorer functional status in the next 4 years [START_REF] Pruchno | A Longitudinal Examination of the Effects of Early Influences and Midlife Characteristics on Successful Aging[END_REF].

Cognitive functioning is also well documented as major underpinnings of functional status [START_REF] Burton | Cognitive Functioning and Everyday Problem Solving in Older Adults[END_REF][START_REF] Hering | Prospective Memory Is a Key Predictor of Functional Independence in Older Adults[END_REF][START_REF] Gross | Cognitive Predictors of Everyday Functioning in Older Adults: Results From the ACTIVE Cognitive Intervention Trial[END_REF]. Indeed, as noted earlier, BADLs and even more so IADLs require the involvement of cognitive resources to remember, plan, focus, read or count during everyday activities realization. Notably, better performance with IADL has been shown to be associated with better prospective memory (i.e., remember and execute delayed intentions) [START_REF] Pruchno | A Longitudinal Examination of the Effects of Early Influences and Midlife Characteristics on Successful Aging[END_REF], better inductive reasoning (i.e., find a common rule from several examples) [START_REF] Gross | Cognitive Predictors of Everyday Functioning in Older Adults: Results From the ACTIVE Cognitive Intervention Trial[END_REF] and better vocabulary [START_REF] Burton | Cognitive Functioning and Everyday Problem Solving in Older Adults[END_REF]. To note, cognitive functioning in older adults is usually assessed with specialized tools providing a global measure of cognition (such as the DRS-2 [START_REF] Jurica | DRS-2 dementia rating scale-2: professional manual[END_REF]) or with specific measures of cognitive functions sensitive to aging such as executive functioning (e.g., the FAB [START_REF] Dubois | The FAB: A frontal assessment battery at bedside[END_REF]).

Similarly, there is ample evidence for a positive relationship between poorer physical aptitude (often called physical frailty [START_REF] Park | Screening Value of Social Frailty and Its Association with Physical Frailty and Disability in Community-Dwelling Older Koreans: Aging Study of PyeongChang Rural Area[END_REF][START_REF] Fried | Frailty in Older Adults: Evidence for a Phenotype[END_REF]) and higher-level ADL loss [START_REF] Shimada | Incidence of Disability in Frail Older Persons With or Without Slow Walking Speed[END_REF][START_REF] Makino | Structural analysis of impact of physical, cognitive and social status on the incidence of disability in community-dwelling people aged ≥75 years[END_REF][START_REF] Seidel | Limitations in physical functioning among older people as a predictor of subsequent disability in instrumental activities of daily living[END_REF][START_REF] Poli | Frailty is associated with socioeconomic and lifestyle factors in community-dwelling older subjects[END_REF]. Indeed, muscle strength, balance, vision, or hearing, are commonly decreasing with age, which may lead to serious ADL limitations. For instance, an impaired balance renders the gait uncertain (i.e., with possible negative outcomes like falls or even sickness), which, in turn, may cause a person to avoid or reduce their mobility [START_REF] Owsley | Mobility impairment and its consequences in the elderly[END_REF].

Taken together, these findings stress that the ADL decline with aging is multi-determined and that the understanding of managing aspects of physical and cognitive functioning into ADLs is likely to be a fruitful way for giving insight into everyday functioning in later life.

Relationships between Cognitive and Physical functioning to perform ADLs in aging

Beside their independent influence over everyday functioning, there is growing evidence suggesting that cognitive and physical functioning tend to be closely intertwined in late adulthood. Notably, in studies using dual tasks [27][START_REF] Poole | Motor-Cognitive Neural Network Communication Underlies Walking Speed in Community-Dwelling Older Adults[END_REF][START_REF] Wollesen | A taxonomy of cognitive tasks to evaluate cognitive-motor interference on spatiotemoporal gait parameters in older people: a systematic review and meta-analysis[END_REF] or even naturalistic tasks like planning a route while walking [START_REF] Taillade | Age-related wayfinding differences in real large-scale environments: detrimental motor control effects during spatial learning are mediated by executive decline?[END_REF] it is observed that older adults tend to "prioritize" sensorimotor processing over cognitive processing. In other words, when dual "cognitive-sensorimotor" abilities are necessary, for instance when walking while reading a map, older adults tend to reduce their performance on the cognitive task (i.e., planning) to maintain their sensorimotor performance (i.e., walking) [START_REF] Taillade | Age-related wayfinding differences in real large-scale environments: detrimental motor control effects during spatial learning are mediated by executive decline?[END_REF]. Besides, some authors even argue that walking in old age is per se a dual task [START_REF] Wollesen | A taxonomy of cognitive tasks to evaluate cognitive-motor interference on spatiotemoporal gait parameters in older people: a systematic review and meta-analysis[END_REF]. This age-related condition directly impacts ADLs which require simultaneous coordination of physical and cognitive functions.

The age-related changes in the coordination of physical and cognitive functions can be related to the issue of compensation: a strategy responding to functional decline in old age that is described by the model of selective optimization with compensation (SOC) [START_REF] Baltes | Psychological perspectives on successful aging: The model of selective optimization with compensation[END_REF]. Applied to the situation of gait and balance loss in later life, compensation means that due to a significantly reduced physical aptitude in daily life, older adults are forced to rely more intensively on cognitive resources to conduct ADLs [START_REF] Zhang | Evidence on selection, optimization, and compensation strategies to optimize aging with multiple chronic conditions: A literature review[END_REF]. Notably, the study of Heyl and Wahl [START_REF] Heyl | Managing daily life with age-related sensory loss: Cognitive resources gain in importance[END_REF] tested the involvement of cognitive resources to compensate sensory impairment in the context of ADL tasks. Indeed, these authors found that cognitive resources and behavior-related everyday functioning are more closely related in older adults having sensory impairments as compared to sensory unimpaired counterparts. Authors discussed their results in the light of the SOC model, and suggest the promotion of cognitive training to support everyday life of elders living with sensory impairment.

In this line of research, the present study attempts to provide further evidence for the involvement of cognitive resources in the maintenance of ADLs in physically frail oldest-old adults. The population selected is a sample of cognitively healthy older adults who are divided into two groups on the basis of their performance on tests assessing their physical functioning (in terms of mobility, corporal balance, body mass and sensory functions). The expected results are that older adults having physical disturbances rely more on their cognitive resources to successfully perform ADL tasks for coping with their physical decline. However, as they are cognitively spared, they should accurately self-perceive their limitations for carrying out ADL tasks.

METHODS

Recruitment and final participants

To test our research assumptions, it was essential to include older participants with physical limitations. For this reason, we collaborated with three public home services for elders randomly selected among all the Gironde municipalities with a location criterion as follows: one urban, one semi-urban and one rural location. In France, the level of public financial support for home service is determined by the functional status. As a result, each file of a home-service beneficiary is documented by the results of a geriatric assessment performed by medical consultants, according to the AGGIR scale (gerontological ISO norms for autonomy practiced in France to establish a person's functional status). Within our sample, the individuals presenting a dependency syndrome (GIR-score inferior to 4) were excluded from the study.

Among the remaining individuals, we conducted around a hundred of phone calls and 86 older adults accepted to participate to the study. They underwent a battery of tests. All the interviews were done at the person's home across two sessions. Thirty-four elders were excluded from the study because of their MMSE score (< 27) to avoid pathological cognitive impairment, such as dementia cases. Then, we created our two groups of subjects regarding their physical functioning scores (see below for the calculation of this score). Eventually, the study sample consisted of 52 community-dwelling old adults aged between 73 to 94 years (mean age 82.2 ± 4.7); 9 males and 43 females; they were still autonomous and without cognitive impairment.

Assessment of Physical functioning

Tasks were selected from widely used clinical and research scales for assessing physical functioning and subsequent frailty [START_REF] Guralnik | A Short Physical Performance Battery Assessing Lower Extremity Function: Association With Self-Reported Disability and Prediction of Mortality and Nursing Home Admission[END_REF][START_REF] Bruyère | How clinical practitioners assess frailty in their daily practice: an international survey[END_REF] as follows:

Five Chair Stands (lower body strength): The participant is asked to stand up from a chair five times without using their arms. The time is recorded and the test is scored from 4 -the participant takes less than 11.1 sec to complete the task to 0the participant is unable to perform task.

Static Balance Testing consists of three sorts of standing: side-by-side stand, semi-tandem stand and tandem stand; each of them scored from 4the participant holds the three standing positions for more than 10 sec; to 0the participant did not attempt any standing position.

Timed Get Up and Go Test (agility and dynamic balance). This test consists of rising from a chair, walking three meters, turning around, walking back to the chair, and sitting down. Time in seconds to complete the task is recorded. The task is scored as followed: 1the task is completed in more than 30 sec, 2-the task is completed from 20 to 30 sec, and 3 -the task is completed in less than 10 sec (in this case, mobility is considered normal).

Gait Speed Test corresponds to a timed 4-meter walk. It is scored from 4time is less than 4.82 secto 0the participant was unable to do the walk.

The score from these four tests ranged from 0 to 13 with higher values indicating greater mobility function.

Body mass is also an important component of physical frailty [START_REF] Fried | Frailty in Older Adults: Evidence for a Phenotype[END_REF]. Thus, two indices have been scored based on the Mini-Nutritional Assessment [START_REF] Vellas | The mini nutritional assessment (MNA) and its use in grading the nutritional state of elderly patients[END_REF]. First, the Body Mass Index (BMI), scored from 0 to 3 with higher values indicating higher BMI values. Second, the brachial and calf perimeters are scored from 0 to 2 with higher values indicating higher lean mass values. Summed, the two indices provide a score from 0 to 5, with a higher score indicating a better body mass.

Finally, sensory abilities, particularly visual acuity and hearing were assessed with a three-point Likert-type scale, ranging from 0 to 2 (where 0 corresponds to the highest sensory loss). So, sensory scale provided score ranged from 0 to 4 with higher scores indicating better sensory functions.

So, the score from all test ranged from 0 to 24 with higher values indicating greater physical functioning (see Table 1). From this score, participants were then divided into two subgroups based on their physical performance (see below): participants above the physical functioning score median were considered as 'high physical functioning' (n=26, 5 males and 21 females), whereas other participants were considered as 'low physical functioning' (n=26, 4 males and 22 females). These two groups were equivalent in terms of socio-demographic characteristics, cognitive functioning (including Mini-Mental State Evaluation, MMSE [START_REF] Folstein | Mini-mental state[END_REF]; and Cognitive resource score described below), and cognitive complaint (assessed by Cognitive Difficulties Scale, CDS [START_REF] Mcnair | The cognitive difficulties scale[END_REF]) (see Table 1).

Assessment of cognitive resources

General cognitive functioning: The Dementia Rating Scale-2 (DRS-2) [START_REF] Jurica | DRS-2 dementia rating scale-2: professional manual[END_REF] has been used. It Executive functioning: The Frontal Assessment Battery (FAB) [START_REF] Dubois | The FAB: A frontal assessment battery at bedside[END_REF] has been administrated. It probes several domains including conceptualization, mental flexibility, motor programming, resistance to interference, self-regulation, inhibitory control, and environmental autonomy.

FAB gives a score ranging from 0 to 18 (where 18 is the maximum score).

The cognitive resource measure refers to the sum of scores obtained on each scale (with a maximum score of 162). As indicated in Table 1, the two groups of old participants did not differ for the cognitive resource measures.

Assessment of everyday functioning

Performance based assessment. We administrated the TIADL Tasks [START_REF] Owsley | Timed Instrumental Activities of Daily Living Tasks: Relationship to Cognitive Function and Everyday Performance Assessments in Older Adults[END_REF], composed of five timed tasks that simulate everyday instrumental activities of daily living: communication (finding a telephone number in the telephone directory), finance (finding and counting out correct change of money), cooking (finding and reading the ingredients on three food cans), shopping (finding two specific items in an array of food items) and medicine (finding and reading the directions on medicine containers). Each task is scored as 1completed without errors and within the time limit, 2completed with minor errors, or 3not completed within the time limit, or completed with major errors. Thus, the TIADL gives a score range from 5 to 15, higher scores indicating more difficulties to perform the tasks.

Self-report assessment. We assess a 24-items scale based on ADL and IADL items [START_REF] Lawton | A Research and Service Oriented Multilevel Assessment Instrument[END_REF], where the answer is based on a 5-point Likert-type format varying from 0not at all difficult, to 4very difficult. Then, we selected the 15 IADL items to identify the self-reported IADL score.

Thus, the self-reported IADL gives a score range from 0 to 60, higher scores indicating more complaint about IADL tasks.

Assessment of Self-perceived health. We assess the health-related quality of life using the Short Form-36 (SF-36) questionnaire [START_REF] Ware | The MOS 36-Item Short-Form Health Survey (SF-36): I. Conceptual Framework and Item Selection[END_REF]. This questionnaire consists of 36 items, covering eight dimensions (physical functioning, physical limitations, body pain, general health, vitality, social functioning, limitations due to emotional problems, and mental health) and provides two summary scores, namely physical score and mental score.

Statistical analyses

First, group comparisons (Low Physical Functioning vs. High Physical Functioning) have been performed with the Student's t test procedure on each measures of everyday functioning (Table 2). For these tests, t-values, p-values (with a significance level <.05) and effect sizes (η²) were reported. As our final sample ended up relatively small, we added post-hoc power sensitivity analyses (observed power β) to prevent from Type-I errors (i.e., false positive conclusions) when significance was observed.

Second, global correlations including all participants have been carried out between measures of everyday functioning and cognitive functioning scores (Table 3). Third, to assess the influence of the Cognitive Resource factor on IADL measures, two multiple linear regression analyses were carried out in the two groups independently, with the following statistical design: physical functioning score and cognitive resource score as predictors, and TIADL and selfreported IADL scores as criterion variables. In these four regressions, percentage of the variance explained by the model (Adjusted R²), and unstandardized (B) and standardized (β) regression coefficients for the variables entered into the model were reported (Table 4). Each effect size was computed with η2. All data analysis was performed using SPSS 20.0.

RESULTS

Effects of physical functioning level on everyday functioning (Table 2)

In

Global relationships between the everyday functioning measures and the cognitive resources measures (Table 3)

As illustrated in Table 3, the performance-based and self-reported measures of IADL are significantly related with a positive coefficient (r = .391; p = .004), which means that more difficulties in the TIADL tasks are associated with more reported difficulties in everyday functioning. Similarly, the physical and mental sub-scores of the SF-36 were strongly related with a positive relationship (r = .513; p < .001). Regarding the relationships with cognitive resources, both performance-based and self-reported measures appeared strongly correlated with cognitive resources but with negative coefficients (TIADL: r = -.748; p < .001; self-reported IADL: r = -.483; p < .001), which means that greater everyday difficulties (observed or reported) are associated with lower cognitive resources.

Similarly, physical functioning score emerged negatively correlated with TIADL and selfreported IADL scores (TIADL: r = -.343; p = .013; self-perceived IADL: r = -.533; p < .001), suggesting that better physical abilities are associated with lower observed or reported everyday difficulties.

Impact of cognitive resources on the relation between physical level and ADL (Table 4)

To assess the relative influence of physical functioning and cognitive resources to manage everyday functioning (and highlight a possible compensatory effect of cognitive resources when physical functioning is limited), multiple regression analyses have been performed on each measure of IADL (TIADL; self-reported IADL) (Table 4). The four models appeared significant (Regression 1: F (2, 25) = 6.640; p = .005; Regression 2: F (2, 25) = 6.744; p = .005; Regression 3: F (2, 25) = 19.294; p = <.001; Regression 4: F (2, 25) = 4.948; p = .016). In both groups, when the TIADL score is entered as the dependent variable (Regression 1 and 3), cognitive resources score was a significant predictor, with a negative relationship with the criterion variable, meaning that more cognitive resources are associated with better performance at the TIADL (i.e., lower scores). In the Low PF group, the adjusted R² of the cognitive score coefficient is higher compared to the High PF group, suggesting a stronger importance in variance explanation. In both models, physical functioning score was not a significant predictor. When the self-reported score is entered as the dependent variable, physical score emerged as a significant predictor in the High PF group (Regression 2) with a negative relationship, indicating that lower physical performance is associated with increased complaint about everyday functioning (i.e., higher scores), and cognitive score remained non-significant. In the Low PF group however (Regression 4), physical score appeared non-significant but cognitive score emerged as a significant predictor, with lower cognitive resources associated with an increased complaint about everyday life.

DISCUSSION

Our study is the first to attempt to demonstrate the compensatory role of cognitive resources among physically frail older adults in everyday functioning. For this purpose, we have recruited cognitively healthy elders, who differed only depending on their physical functioning level (Low Physical Functioning vs. High Physical Functioning). Performance-based and selfreported measures of everyday functioning have been collected.

A major result is that cognitively healthy elders exhibited similar performance-based everyday functioning irrespective of their level of physical functioning. In other words, old participants with low physical functioning successfully handled their physical limitations to adequately achieve the IADL tasks. Nevertheless, these participants reported more problems regarding IADL realization and physical health than their counterparts with high physical functioning. This indicated that even if elders with low physical functioning remain able to properly perform IADL tasks, they accurately perceive their physical limitations for carrying out such tasks. These results are fully consistent with our expectations and the literature. Indeed, this result reinforces the critical role of physical functioning [START_REF] Shimada | Incidence of Disability in Frail Older Persons With or Without Slow Walking Speed[END_REF][START_REF] Seidel | Limitations in physical functioning among older people as a predictor of subsequent disability in instrumental activities of daily living[END_REF][START_REF] Owsley | Mobility impairment and its consequences in the elderly[END_REF] and cognitive health [START_REF] Hering | Prospective Memory Is a Key Predictor of Functional Independence in Older Adults[END_REF][START_REF] Gross | Cognitive Predictors of Everyday Functioning in Older Adults: Results From the ACTIVE Cognitive Intervention Trial[END_REF][START_REF] Shimada | Incidence of Disability in Frail Older Persons With or Without Slow Walking Speed[END_REF] in self-reported everyday functioning and physical health. Additionally, our results emphasize the possible incongruence between performance and self-reported measure of IADL in physically frail elders. Indeed, normal cognitive aging studies mostly give evidence for congruence between performance-based and self-reported scores of everyday difficulties [START_REF] Burton | Cognitive Functioning and Everyday Problem Solving in Older Adults[END_REF][START_REF] Bravell | Self-reported activities of daily living and performance-based functional ability: a study of congruence among the oldest old[END_REF], which is perfectly illustrated in our old participants with high physical functioning.

However, when an incongruence between performance and reported IADL (and more precisely an overestimation of difficulties compared to the actual performance) is observed among cognitively healthy elders, while some authors proposed the influence of age [START_REF] Wettstein | Still feeling healthy after all these years": The paradox of subjective stability versus objective decline in very old adults' health and functioning across five years[END_REF][START_REF] Fors | Do actions speak louder than words? Selfassessed and performance-based measures of physical and visual function among old people[END_REF], cognition [START_REF] Suchy | Instrumental activities of daily living among community-dwelling older adults: Discrepancies between self-report and performance are mediated by cognitive reserve[END_REF][START_REF] Fors | Do actions speak louder than words? Selfassessed and performance-based measures of physical and visual function among old people[END_REF] or culture [START_REF] Spitzer | Who is telling the truth? Biases in self-reported physical and cognitive health status of older Europeans[END_REF], this can be an early indicator of physical decline and cognitive compensation process involved. Hence, ADL performance during performance-based assessments probably relies more heavily on cognitive resources than on physical functioning, which is supported by the strong correlation observed between cognitive resources and TIADL scores (irrespective of physical functioning conditions). Such pattern also underlines the limit of performance-based assessments, which exclude individual's chosen routines, strategies and environmental cues that typically facilitate ADL in everyday life [START_REF] Gold | An examination of instrumental activities of daily living assessment in older adults and mild cognitive impairment[END_REF], and suggest the administration of both performance-based and self-reported measures of everyday difficulties.

To sum up, it is clear that the incongruence reported for cognitive healthy elders with low physical functioning deserves to be further studied; notably with regard to the potential role of cognitive functioning as a moderator of physical loss on the performance-based measures of IADL.

Along the line with research by Heyl and Wahl [START_REF] Heyl | Managing daily life with age-related sensory loss: Cognitive resources gain in importance[END_REF] on sensory deficit, we specially addressed the moderating effect of cognitive resources on physical loss during IADL measures.

Regression results highlighted that the influence of cognitive resource on performance-based IADL is significantly modified in respect with the level of physical functioning (i.e., adjusted R² of the Cognitive resources score with TIADL score as a criterion variable). Importantly, the importance of cognitive resources to predict the variation of performance on IADL tasks is significantly increased by a low physical functioning condition compared to the high physical functioning condition. Consequently, the evidence of a compensatory role of cognitive resource in performance-based IADL for physically frail elders is provided from this angle. Additionally, our compensatory assumption is supported by a second major result. The significant predictors of the self-reported IADL measure differ depending on physical functioning level. The regressions analyses revealed that the variation of IADL difficulties reported by participants with low physical functioning is significantly predicted by their level of cognitive resources.

Contrariwise, in participants with high physical functioning, this variation is only predicted by their level of physical functioning. This means that self-reported IADL varies as follows: for the old-old participants with high physical functioning, having physical difficulties (even a few)

is the main reason why they would report IADL problems; however, for older adults with low physical functioning, this is the lack of enough cognitive resources that would predict their selfreported IADL problems. In other words, in the situation of physical loss, cognitively healthy older adults who have the least cognitive resources are acutely aware of the cognitive effort they exert to achieve nominal ADL performance. Thus, the additional cognitive effort for maintaining IADL performance in physically frail older adults is probably experienced as more effortful among those having less cognitive resources than those with higher ones. This result goes further the recently published paper showing that cognitive status indirectly affects physical aging in the apparition of disability [START_REF] Makino | Structural analysis of impact of physical, cognitive and social status on the incidence of disability in community-dwelling people aged ≥75 years[END_REF], since it proposed an explanation for this indirect effect, i.e., compensation, as suggested in the SOC Model [START_REF] Heyl | Managing daily life with age-related sensory loss: Cognitive resources gain in importance[END_REF][START_REF] Baltes | Psychological perspectives on successful aging: The model of selective optimization with compensation[END_REF].

Some limitations of this study can be noted. The first is related to the modest size of the sample included in the study (even if we did prevent from false positive conclusions by running power sensitivity analyses). With a larger sample size, statistical power of our results relative to compensatory effect could be reinforced. Second, some studies [START_REF] Hering | Prospective Memory Is a Key Predictor of Functional Independence in Older Adults[END_REF][START_REF] Gross | Cognitive Predictors of Everyday Functioning in Older Adults: Results From the ACTIVE Cognitive Intervention Trial[END_REF] found differential impact of different cognitive functions on self-reported and performance-based assessment of everyday functioning, while we used a global measure of cognitive resources. Further works could investigate the relations between specific cognitive functions (e.g., mental flexibility or inhibitory control) and measures of everyday functioning for defining more finely which cognitive resource is involved in the cognitive compensation in situations of physical loss.

Third, we used scores at the TIADL tasks to measure IADL performance (as suggested by the designer of the test [START_REF] Owsley | Timed Instrumental Activities of Daily Living Tasks: Relationship to Cognitive Function and Everyday Performance Assessments in Older Adults[END_REF]), but finer grain measure of the performance, such as the time used or the type of error (as adapted in [START_REF] Hering | Prospective Memory Is a Key Predictor of Functional Independence in Older Adults[END_REF]) could give further insight of compensation strategies involved in everyday life.

To conclude, our study is the first to highlight a compensatory role of cognitive resources in response to physical loss, among older-old adults with normal cognitive aging.

Several of our results support this compensation view and give some insights on the intertwining with aging between physical and cognitive functioning to move forward the field of aging and everyday functioning. Additionally, underpinning Poli et al.'s recommendations [START_REF] Poli | Frailty is associated with socioeconomic and lifestyle factors in community-dwelling older subjects[END_REF] in their recent study, our findings stress the need for cognitive and psychosocial interventions for elders with physical loss, in order to counteract their vulnerability induced by their continuous cognitive efforts in everyday life. In doing so, we would further support the preservation of older adults' autonomy and promote successful aging.

  assesses five cognitive domains, including attention (e.g., forward and backward digit span, ability to follow commands), initiationperseveration (semantic fluency, motor fluency and perseveration), abstraction (conceptualization from verbal and non-verbal stimuli), visualconstructional abilities (copy of geometric figures and signature writing) and verbal as well as non-verbal memory (recall and recognition). This scale gives a score between 0 and 144 (where 144 is the best score).

Table 1 :

 1 Characteristics of the two groups of older participants according to the level of physical functioning (High vs. Low Physical Functioning)

		Physical Functioning
		Low	High	Group	Effect size (η²)
		Mean (SD)	Mean (SD)	comparison	Obs. power (β)
	Age	83.12 (.88)	81.12 (.95)	t(50) = 1.54; p = .13 η² = 0.045
	Gender (% female)	80.77	84.61	t(50) = .36; p = .72	η² = 0.002
	Education years	9.81 (.45)	8.96 (.49)	t(50) =1.27; p = .21	η² = 0.031
	Family Status (% married)	23.08	19.23	t(50) = .333; p = .74 η² = 0.002
	MMSE (max. = 30)	28.26 (0.24)	28.35 (.25)	t(50) = -.26;	η² = 0.001
				p = . 78
	Physical functioning score	13.40 (.77)	20.62 (.32)	t(
	(max. = 24)

50) = -8.67; p < .0001 η² = 0.600 β = 1.000

	Cognitive Resources score	146.24 (3.31)	150.30 (1.68) t(50) = -1.809;	η² = 0.061
	(max. = 174)			p = . 28
	CDS (max. =144)	42.95 (4.26)	34.33 (3.68)	t(50) = 1.53;	η² = 0.045
				p = . 13
	Note. SD=standard deviation; MMSE=Mini-Mental Status Exam; CDS=Cognitive Difficulties Scale.

  terms of performance-based assessment, the Low and High Physical Functioning groups had

	nearly similar performance on the TIADL test (t(50) = .968 ; p > .300; η² = 0.018). Interestingly,
	in terms of self-report assessment, self-reported difficulties to perform everyday activities (self-
	report IADL score: t(50) = 3.27; p = .002) are higher for the Low Physical Functioning group
	than for the High Physical Functioning group. The strong observed power (β = 89.5%) and
	effect size (η² = 0.177) values suggest that the difference between the two groups is not a false
	positive.
			Physical Functioning
			Low	High
			Mean (SD)	Mean (SD)	Group	Effect size (η²)
					Comparison	Obs. power (β)
	TIADL		6.15 (0.466)	5.65 (0.221)	t(50) = 0.968;	η² = 0.018
	(max. = 15)				p = .337
	Self-reported IADL	14.46 (1.72)	7.50 (1.25)	t(50) = 3.275;	η²	=	0.177
	(max. = 60)				p = .002	β = 0.895
	SF-36	physical	37.28 (4.04)	57.24 (3.45)	t(50) = -3.76;	η²	=	0.261
	(max. = 100)			p < .001	β = 0.958
	SF-36 mental	55.07 (4.56)	63.41 (4.36)	t(50) = -1.32;	η² = 0.034
	(max. = 100)			p>.100
	Note. SD=standard deviation; IADL=Instrumental Activities of Daily Living; SF-36=Short Form-36.

Concerning the assessment of self-perceived health, the two groups of participants did not differ in terms of self-perceived mental health, i.e., SF-36's mental sub-score (t(50) = -1.32 ; p > .100; η² = 0.034). By contrast, compared to the High Physical Functioning group, the Low Physical Functioning group performed lower on the SF-36's physical sub-score (t(50) = -3.76; p < .001), indicating a decreased self-perceived physical health, which does not seem to be due to a false positive result (η² = 0.261 ; β = 95.8%). Table 2: Performance-based and self-reported measures of Everyday functioning for the two groups of older participants (High vs. Low Physical Functioning group).

Table 3 :

 3 Inter-correlations between measures of everyday functioning and cognitive andphysical measures for all participants group.

		TIADL	Self-report	SF-36	SF-36	Cognitive	Physical
			IADL	physical	mental	Resources	Functioning
	TIADL	-	.391**	.042	-.058	-.748***	-.343*
	Self-reported IADL		-	-.405**	-.220	-.483***	-.533**
	SF-36 physical			-	.513***	.062	-.440**
	SF-36 mental				-	.253	.074
	Cognitive Resources					-	.329*
	Notes. * p < .05, *** p < .001. Significant correlations are bolded

Table 4 :

 4 The unstandardized (B), standardized regression coefficients (β), and percentage of variance explained (Adjusted R²) for the variables entered into the four models

	Variables	B	SE B	β	p	Adj R² (%)
	Regression 1 : High PF group -TIADL as criterion variable
	Physical	.002	.115	.002	.989	3.6
	Cognitive	-.080	.022	-.605	.001	27.5
	Regression 2 : High PF group -self-reported IADL as criterion variable
	Physical	-2.071	.648	-.533	.004	26.3
	Cognitive	-.174	.124	-.233	.175	5.2
	Regression 3 : Low PF group -TIADL as criterion variable
	Physical	-.103	.084	-.169	.232	16.2
	Cognitive	-.100	.019	-.711	< .001	43.2
	Regression 4 : Low PF group -self-reported IADL as criterion variable
	Physical	-.281	.422	-.126	.513	6.0
	Cognitive	-.254	.098	-.488	.017	18.0

Notes. SE: Standard Error; * p < .05, *** p < .001. Significant predictors are bolded

Acknowledgments

The authors are very grateful to the services of UDCCAS Gironde that allowed them access to the elderly.

Funding This work was supported by the National French Institute of Informatics and Mathematics (Inria), the Public funds Conseil Régional d'Aquitaine, CNSA (Caisse Nationale de la Solidarité pour l'Autonomie), Conseil Départemental de la Gironde, and CARSAT Aquitaine (Caisse d'Assurance Retraite et de la Santé au Travail) and the Fondation pour la Recherche Médicale, (grant number : DOC20161136217; PI: Sauzéon).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Compliance with ethical standards

Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.

Statement of human and animal rights

According to Helsinki declaration, approval was sought and obtained from the ethics committee of the Bordeaux University.

Informed consent All participants provided a written consent form prior to participation in the study.


David Mitton

Edison Zapata

Hélène Follet

come

  Mechanical loading at the organ level: whichconsequences for bones? Hélène Follet, David Mitton, Edison Zapata To cite this version:Hélène Follet, David Mitton, Edison Zapata. Mechanical loading at the organ level: which conse-quences for bones?. Osteoporosis International, Springer Verlag, 2014, Eighth Bone Quality SeminarProceedings 2013, 25 (Supplément 3), pp.S468-S470. 10.1007/s00198-014-2681-x. hal-02353938