Aged TiO2-Based Nanocomposite Used in Sunscreens Produces

Article pubs.acs.org/est

Aged TiO2‑Based Nanocomposite Used in Sunscreens Produces Singlet Oxygen under Long-Wave UV and Sensitizes Escherichia coli to Cadmium Catherine Santaella,*,†,‡,§,∥ Bruno Allainmat,†,‡,§,∥ France Simonet,⊥ Corinne Chanéac,∥,# Jérome Labille,∥,∇ Mélanie Auffan,∥,∇ Jérome Rose,∥,∇ and Wafa Achouak†,‡,§,∥ †

CEA, IBEB, Laboratory of Microbial Ecology of the Rhizosphere and Extreme Environments, Saint-Paul-lez-Durance, F-13108, France ‡ CNRS, UMR 7265 Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance, F-13108, France § Aix Marseille Université, BVME UMR7265, Marseille, F-13284, France ∥ GDRi iCEINT, International Consortium for the Environmental Implication of Nanotechnology, CEREGE, F-13545 Aix-en-Provence, France ⊥ IRCELYON, UMR 5256 CNRS- Lyon 1 Univ, Villeurbanne, F-69100, France # Chimie de la Matière Condensée, UMR7574, Collège de France, Université de Jussieu, Paris, F- 75231, France ∇ CNRS, Aix-Marseille Université, CEREGE UM34, UMR 7330, 13545 Aix en Provence, France S Supporting Information *

ABSTRACT: TiO2-based nanocomposite (NC) are widely used as invisible UV protectant in cosmetics. These nanomaterials (NMs) end in the environment as altered materials. We have investigated the properties of T-Lite SF, a TiO2−NC used as sunscreen, after weathering in water and under light. We have examined the formation of ROS and their consequences on cell physiology of Escherichia coli. Our results show that aged-T-Lite SF produced singlet oxygen under low intensity long wave UV and formed hydroxyl radicals at high intensity. Despite the production of these ROS, T-Lite SF had neither effect on the viability of E. coli nor on mutant impaired in oxidative stress, did not induce mutagenesis and did not impair the integrity of membrane lipids, thus seemed safe to bacteria. However, when pre-exposed to T-Lite SF under low intensity UV, cells turned out to be more sensitive to cadmium, a priority pollutant widely disseminated in soil and surface waters. This effect was not a Trojan horse: sensitization of cells was dependent on the formation of singlet oxygen. These results provide a basis for caution, especially on NMs that have no straight environmental toxicity. It is crucial to anticipate indirect and combined effects of environmental pollutants and NMs.

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INTRODUCTION Nanotechnology-enabled products have invaded the life of consumers worldwide with 1628 identified products in 30 countries by the end of 2013 (www.nanotechproject.org). The market of manufactured nanoparticles rises continuously. Interestingly, the public perception of nanotechnologies has evolved from negative opinions to weighing up of benefits with risks (www.nanotechproject.org). Nano titanium dioxide is the largest production among nanomaterials (NMs), with high variance in the estimations of manufactured volumes: 7800−38 000 t/year in the U.S.,1 550−5500 t/year worldwide,2 and 50 400 t/y worldwide in 2010.3 Nano-TiO2 has photocatalytic properties. Under UV illumination, the nanoparticles produce reactive oxygen species (ROS) that interact with organic matters in self-cleaning materials and surfaces, air and water treatment. When the surface of the particle is protected with inorganic and/or organic layers to preclude the formation of © 2014 American Chemical Society

ROS, nano-TiO2 absorb UV and is used to protect skin and surfaces from UV-damages in cosmetics, paints, and textiles. According to manufacturer’s declarations, a large part of the nano-TiO2 production (70−80%) is stated be used as UVprotector in cosmetics, including sunscreens.1 The nanosized TiO2 in food products and personal care products may release as much as 16 mg of nanosized TiO2 per individual per day to wastewater.2 The uses of manufactured NMs lead inevitably to their dissemination in the environment. For nano-TiO2 used in cosmetics, the expected scenario anticipates an end up in wastewater, surface water, exposure of landfills, and in soils and Received: Revised: Accepted: Published: 5245

January 14, 2014 April 2, 2014 April 3, 2014 April 3, 2014 dx.doi.org/10.1021/es500216t | Environ. Sci. Technol. 2014, 48, 5245−5253

Environmental Science & Technology

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surface water after sewage treatment.3 The consequences are not yet totally assessed. The speciation of NMs released in the environment will differ according to the stage level of the value chain, as NMs are modified before their incorporation in commercial products. Therefore, during the usage stage, nanocomposites more than pristine NMs are more likely to be released. NMs that will be released will differ from the native manufactured ones, as they will be weathered and aged during subsequent interactions with the environment.4 Aging or weathering of NMs refers to several processes such as changes in size, coating, shape, surface chemistry, and reactivity. Aging of NMs can change the interactions with living organisms. Hence, aging in simulated groundwater of nanoscale zerovalent iron (NZVI), a redox-active nanomaterial used to remediate groundwater pollution, changes average oxidation states.5 Aged/oxidized NZVI particles have lower redox activity that correlates with a decrease in the cytotoxicity of ion nanoparticles to Escherichia coli.6 Upon storage, citrate- and poly(vinylpyrrolidone)-stabilized silver nanoparticles release silver ions, which increases the toxicity to human mesenchymal stem cells.7 Zinc oxide nanopart icles aged in kaolin suspension rapidly convert via destabilization/dissolution mechanisms to Zn2+ innersphere.8 Aging changes the crystallinity and the morphology of Dumbbell-shaped nanocomposites used in biomedicine.9 Recently, the toxicity of a TiO2-based nanocomposite (TiO2−NC) used in sunscreens, was assessed on varied organisms, after weathering/aging and compared to pristine nano-TiO2. This nanocomposite consists in a rutile TiO2 core and is coated with Al(OH)3 and polydimethylsiloxane (PDMS) layers to avoid photo-oxidation reactions and the formation of reactive oxygen species (ROS). After aging in water under light, a stable colloidal phase (50−700 nm) is formed. The organic PDMS is desorbed and oxidized but the layer of Al(OH)3 is still at the surface of the composites.10,11 The eco-impact of this weathered TiO2−NC is assessed in earthworms,12,13 a leguminous plant,14 an aquatic organism,15 a bacterium16 and in human cells.17 This weathered TiO2−NC shows no toxicity to human cells up to 100 mg/L but alters the antioxidative and apoptotic activity in worms, decreases growth and reproduction in Daphnia magna, induces oxidative stress in the root system of Vicia faba, and increases genotoxicity at high dose in Salmonella typhimurium. In this study, we investigated the impact of aged-TiO2 NC on the model bacterium, Escherichia coli, which is a bioreporter in ecotoxicology.18 The photocatalytic properties and the toxicity of aged T-Lite SF was checked against those of nanoparticles made of pristine rutile nano-TiO2 (n-TiO2) and gibbsite nanoAl(OH)3 (n-Al(OH)3) NMs, on wild type E. coli cells and mutants altered in oxidative stress. We studied the influence of illumination, consistent with natural exposure, and of medium, using water to favor interactions, but also solutions controlled in pH and salt composition. We also focused on the impact of the successive exposure of cells to aged-TiO2 NC and cadmium, a priority pollutant widely disseminated in soil and surface waters. Our results provide a basis for caution, especially on NMs that do not show effective and straight environmental toxicity. It is crucial to understand and anticipate indirect and combined effects of environmental pollutants and NMs.

Article

MATERIALS AND METHODS

Products, Bacteria, and Incubation Conditions. Aged TiO2 nanocomposite (NC) from T-Lite SF (BASF) was prepared according to Labille et al.10 Nanoparticles of gibbsite Al(OH)3 and pristine TiO2 (rutile) were prepared by soft chemistry as previously described.19,20 The nanomaterials (NMs) were characterized by dynamic light scattering (DLS) and electrophoretic mobility (Zeta Nanosizer, Malvern), and by transmission electron microscopy. Suspensions of rutile n-TiO2 and n-Al(OH)3 fitted the Ti or Al concentration of the aged TLite SF nanocomposite. The stable colloid phase of aged T-Lite SF at 106 mg/L contained 77 mg/L of Ti and 5 mg/L of Al.10 Supporting Information Table S1 summarized the physicochemical parameters of the NMs. Supporting Information Figure S1 shows images of the NMs by transmission electron microscopy. Bacteria were exposed to NMs and aged T-Lite SF, in ultrapure water (UPW, pH 5.5), KCl (9 g/L, pH 6.8), and Modified Davis medium (MDM) (pH 6.0, K2HPO4 (0.7 g/L), KH2PO4 (0.2 g/L), (NH4)2SO4 1 g/L, citrate Na 0.5 g/L, MgSO4 0.1 g/L). Light exposure was implemented under long wave UV 365 nm, 0.365 mW/cm2 (low UVA intensity exposure) or 5 mW/ cm2 (high UVA intensity exposure). Bacterial strains used in this work are Escherichia coli WT strain QC1301 (MG1655), and mutants sodAsodB (QC 2472) and katEkatG (QC 2476) from Dr. Touati collection. Cell-Based Bioassays. Cells were grown in Luria−Bertani (LB) medium at 37 °C, up to the middle exponential phase (OD600 nm 0.4−0.5), centrifuged at 5.9g, 4 °C, 20 min, and washed twice in UPW at 4 °C. Typically, 106−107 cells were incubated with NMs at 37 °C in black 96-wells hydrophobic polystyrene plates (Nunclon, Corning) under lateral shaking. Cells and NMs were incubated with lateral shaking in the appropriate medium for 1 h or 15 h at 37 °C under dark conditions or under UVA exposure. For the experiments with cadmium ions, cells were first incubated with NMs under 0.365 mW·cm−2 or in the dark for 1 h at 37 °C under lateral shaking. Then, a solution of cadmium nitrate (0−30 μM) was added and the plates were further incubated at 37 °C for 15 h under dark conditions and lateral shaking. Cell Viability. Serial 10-fold dilutions of cell suspensions were spotted on LB-agar plates. Colony-forming unit were counted.6 We calculated the mean of CFU/mL from three independent biological replicates of cell incubations with NMs. ROS Measurements. Superoxide was detected by reduction of (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) to formazan, as described in.21 Nanomaterials ([Ti] = 7 mg/L or [Al] = 0.5 mg/L) and XTT 100 μM were incubated in water under UVA (365 nm, 0.365 mW/cm−2) at 25 °C. The absorbance at 470 nm was monitored overtime on an UV/visible spectrophotometer (VICTOR2). The production of singlet oxygen was monitored using Singlet Oxygen Sensor Green dye (SOSG, Molecular Probe) as previously described.22 SOSG reacts with 1O2 to produce an endoperoxide that fluoresces with excitation at 488 nm and emission at 528 nm. SOSG (5 μM) and nanomaterials ([Ti]13 = 7 mg/L or [Al] = 0.5 mg/L) were incubated in ultrapure water, KCl or MDM under UVA (365 nm, 0.365 mW/cm−2 or 5 mW/cm−2) for 1 h at 25 °C. 5246

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Electron Paramagnetic Resonance Spectroscopy (EPR). Singlet oxygen was detected by spin-trapping and EPR using 2,2,6,6-Tetramethylpiperidine (TEMP, SIGMA) as described.23 Aged T-Lite SF ([Ti] = 7 mg/L and TEMP (0.1 M) were illuminated under UVA (365 nm, 0.365 mW/cm−2 or 5 mW/cm−2) for 1 h at 25 °C. For quenching of ROS, sodium azide (1.5 M) or DMSO (10% v/v) was added before illumination. The spectra were recorded before and after exposure to long wave UV on a Bruker ER-200. Frequency, 9.43 GHz; microwave power, 2.03 mW; scan width, 65 G; resolution, 2048; receiver gain, 1 × 104; conversion time, 5.12 ms; time constant, 1.28 ms; sweep time, 10.49 s; scans, 2; modulation frequency, 100 kHz. Mutagenesis Assay. E. coli cells were grown up to the middle of exponential phase and inoculated to LB, washed with KCl and resuspended in KCl (106 cell/mL). Nanomaterials were added ([Ti] = 7 mg/L or [Al] = 0.5 mg/L) and the suspensions were illuminated under long wave UV (365 nm, 0.365 mW/cm−2) for 3 h. One mL was resuspended in 10 mL of LB and grown up to the end of exponential phase. Cell populations were enumerated on LB and LB supplemented with rifampicin 100 μg/mL. TBA Assay. Peroxidation of lipids yields malondialdehyde that can be detected by reaction with thiobarbituric acid (TBA). E. coli cells grown in LB to the middle of exponential phase, washed with water and resuspended in water at DO600 nm = 1. Cells were incubated with NMs ([Ti] = 7 mg/L or [Al] = 0.5 mg/L) under UVA (365 nm, 0.365 mW/cm−2) for 1 h at 25 °C. The TBA assay was performed as described in Lyon et al. 2008. Controls with the NMs alone were run to check potential interferences between the NMs and TBA (see Supporting Information Figure S2). One unit of OD532 nm is 0.4 mM MDATBA-adduct. Scanning Electron Microscopy. E. coli cells in exponential phase (106 cell/mL) were incubated with NMs ([Ti] = 7.7 mg/ L) in ultrapure water for 2 h under long wave UV (365 nm, 5 mW/cm−2). Samples were fixed in glyceraldehyde 4% (v/v), cacodylate 0.2 M (pH 7.4) for 1.5 h and stored at 4 °C. Then, samples were washed with cacodylate buffer and dehydrated through a graded series of ethanol−water mixtures up to 100% ethanol. They were finally sputter-coated with gold and observed by using a FEI ESEM model XL 30 scanning electron microscope. Statistic Analyses. Statistical analyses were conducted using STATGRAPHICS Centurion XVI.I. One way and multifactor analyses of variance (ANOVA) were performed on data. In figures, error bars are standard deviation or confidence intervals from the multirange tests.

Figure 1. Formation of ROS by aged T-Lite SF 10 mg/, pristine TiO2 rutile (7.7 mg/L) and Al(OH)3 (0.5 mg/L) under long wave UV at 25 °C. Incubation media were ultrapure water (UPW), KCl, minimum Davis medium (MDM). Errors bars are standard deviations. Letters indicate the treatments that were statistically different from the control (p < 0.05). (a) Fluorescence of SOSG with NMs in UPW (dark bars), KCl (light gray bars) and in MDM (white bars). (b) Fluorescence of APF in interaction with NMs in UPW (dark bars) and in KCl (light gray bars). (c) Detection of O2•− by XTT. Different letters indicate significant differences (p < 0.05).

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RESULTS Aged TiO2-Based Nanomaterials Used in Sunscreens Produces Singlet Oxygen and Hydroxyl Radicals Under UVA Illumination Despite the Al(OH)3 Coating. Based on singlet oxygen sensor green (SOSG) assay, aged T-Lite SF and rutile n-TiO2 ([Ti] = 7.7 mg/L) formed singlet oxygen (1O2) in water and in KCl under low intensity UVA light (Figure 1a). 1 O2 reacts selectively with SOSG, to form a fluorescent endoperoxide.24 As the fluorescence intensity decreases with pH, the intensity of the signals cannot be compared between different media for a given nanomaterial. In MDM, only TiO2 produced some 1O2. The phosphate, present in MDM, is known to have strong quenching activity on 1O2.25

The formation of hydroxyl radicals (HO•) by exposure of nanomaterials under UVA was monitored using 3′-p-aminophenyl fluorescein (APF). APF reacts with 1O2, HO•, superoxide (O2•−), and H2O2, but the fluorescence from reaction with HO• is a thousand fold superior to that of the probe with other ROS (Invitrogen.com). Among the NMs tested, only nano-TiO2 produced some HO• under low intensity UVA exposure (Figure 1b) in UPW and KCl. Superoxide radical (O2•−) production was monitored using the reduction of tetrazolium dye, XTT, by O2•− to a soluble formazan that can be readily quantified in solution.26 Under 5247



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The production of 1O2 can generate oxidative reactions of cellular macromolecules, such as protein oxidation, and DNA mutations, resulting in cell death.27 In order to investigate the ecotoxicity of aged T-Lite SF on E. coli, cells were harvested in the middle of the exponential phase. In that physiological state, some genes coding for antixoxidative stress, such as Mnsuperoxide dismutase (sodA) and catalase (katG) are inducible. E. coli cells were exposed to aged T-Lite SF, pristine nTiO2- and n-Al(OH)3 in water, KCl and phosphate buffer MDM, under UVA. The populations of culturable cells were estimated after serial dilution in pure water and plating on LA. Aged T-Lite SF had no significant effect (p > 0.05) on E. coli survival in the [Ti] range 0−77 mg/L under low UV intensity whatever the medium of incubation used. In the same way, Al(OH)3-NMs did not alter significantly (p > 0.05) cell survival in the [Al] range 0−5 mg/L. However, pristine TiO2 proved toxic from 7.7 mg/L in UPW and KCl, but the toxicity was significantly reduced in MDM (Figure 3).

low intensity UVA exposure, T-Lite SF in UPW produced no O2•− (Figure 1) as already shown by Auffan et al.,11 unlike pristine TiO2. No ROS (1O2, O2•−, HO•, H2O2) was observed for nanoAl(OH)3, under UVA illumination whatever the medium of incubation (Figure 1). As expected, pristine rutile nano-TiO2 produced the whole set of ROS (1O2, O2•−, HO•) whatever the dispersion medium. We used electron paramagnetic resonance spectroscopy (EPR) and spin trapping to confirm the formation of ROS by T-Lite SF under low intensity UVA and to further analyze ROS produced under higher light intensity. The spin trapping 2,2,6,6-Tetramethylpiperidine (TEMP) reacts with 1O2 to yield the free radical 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TEMPO that has a characteristic spectrum with three lines of equal intensity.23 Under low UVA exposure, T-Lite SF formed 1O2 as revealed by the detection of the triplet of TEMPO (Figure 2a). Addition of NaN3 quenched 1O2 (Figure

Figure 3. Survival of E. coli expressed as LOG(N/N0) after exposition to TiO2 -nanocomposite (T-Lite), Al(OH) 3 and pristine TiO2 nanomaterials (NMs) in ultra pure water (UPW), KCl (9 g/L) and modified Davis medium (MDM). Cells in exponential phase (106 cell/ mL) were incubated with the NMS for 15 h at 37 °C under UVA 365 nm, 0.365 mW/cm2. From foreground to background [Ti] = 0, 0.7, 7.7, and 70 mg/L or [Al] = 0. 0.05, 0.5, and 4.5 mg/L. Asterisks (*) denote a significant difference from the control (no nanoparticles added) at the 95% confidence level.

Figure 2. Detection of singlet oxygen based on spin trap TEMP. Aged T-Lite SF (10 mg/L) and TEMP (0.1M) in UPW were exposed to long wave UV (365 nm) for 1 h. (a) T-Lite SF and TEMP after illumination under 0.365 mW/cm−2. (b) T-Lite SF after 0.365 mW/ cm−2 illumination with TEMP as a spin trap and NaN3 (0.3 M) as single oxygen quencher. (c) T-Lite SF after 5 mW/cm−2 illumination for 1 h in the presence of TEMP. (d) T-Lite SF after illumination under 5 mW/cm−2 for 1 h in the presence of TEMP and DMSO as HO• quencher. (e) T-Lite SF and TEMP before illumination. (f) TEMP illuminated with 5 mW/cm−2.

Singlet oxygen induces and inactivates major antioxidant enzymes, such as superoxide dismutase (sodA) and catalase (katE) that suppress oxidant species such as superoxide and peroxides.27 E. coli superoxide dismutase and catalase genes are members of two major oxidative stress regulons, the regulons OxyR and SoxRS.28 SoxRS regulon includes genes such as Mn superoxide dismutase (sodA) that suppresses superoxide radicals. OxyR regulon includes genes as catalases (katG, ahpC and ahpF) involved in resistance to peroxides. Therefore, we have investigated the impact of the NMs on mutants impaired in superoxide dismutase A and B (sodAsodB) and catalase E and G (katEkatG). Mutants affected in oxidative stress were not altered in their survival by the interactions with T-Lite SF and n-Al(OH)3 (Figure 4) under low UVA exposure. Pristine n-TiO2 induced strong reduction of cell survival for mutants affected in oxidative stress. E. coli Cells and TiO2-Based NMs Interact Unevenly. The interactions between cells and nanomaterials were investigated using scanning electron microscopy (Figure 5).

2b) and restored the weak triplet observed in the spectrum of T-Lite SF and TEMP before exposure to light (Figure 2e) or TEMP alone illuminated under UVA (Figure 2f). Under high UVA intensity (5 mW/cm2), T-Lite SF did not generate the signal of TEMPO suggesting that no 1O2 was produced (Figure 2c). However, HO• is likely to react with the nitroxyl group of TEMPO and silent the spectrum. Addition of DMSO as a quencher of HO•, restored the spectrum of TEMPO (Figure 2d). Thus, T-Lite SF formed only 1O2 under low UV intensity and 1O2 and HO• under high UV intensity. ROS Produced by TiO2 Nanocomposite Used in Sunscreens Have Neither Impact on the Survival of E. coli Cells nor on Mutants Affected in Oxidative Stress. 5248



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oxidation of guanine but also from 1O2 reactivity to cell components other than genetic material, such as cell membranes.31 The treatment of E. coli strains with compounds that generate 1O2 induces cell death and mutagenesis, detected with the induction of rifampicin resistant colonies.32 The mutation rate toward rifampicin resistance is a sensitive assay for mutational events involving base substitutions.33 Peroxidation of lipid fatty acids yields malondialdehyde that can be monitored with thiobarbituric acid. Incubation of cells in the presence of aged T-Lite SF under low UVA intensity did not induce lipid peroxidation (Supporting Information Figure S2). Mutation rate of cells incubated with T-Lite SF under UVA, were not different from the rate of control cells: mutation rate was 4.9.10−7 ± 2.7 × 10−7 for E. coli cells and 3.6. 10−7 ± 2.4. 10−7 for cells incubated with T-Lite SF (error bars are confidence intervals). Thus, singlet oxygen generated by aged T-Lite SF under low UVA intensity yielded neither detectable lipid peroxidation nor mutagenesis. Cell Pre-Exposed to TiO2−NC Under UVA Become Sensitive to Cadmium Due to Singlet Oxygen Production. Cells pre-exposed to aged T-Lite SF under UVA showed higher sensitivity to cadmium as compared to cells exposed to UVA without NC (Figure 6a). Single hour incubation with aged T-Lite SF reduced by half and one log the survival of cells further exposed to concentration of Cd2+ as low as 3 μM and 10 μM, respectively. Thus, the exposure of cells to aged T-Lite SF did not impair basic and vital functions but altered some operational mechanisms that help the cell to overcome cadmium toxicity. When the whole experiment was performed in darkness (i.e., pre-exposure to NMS and incubation with Cd2+), the survival of cells subjected to aged T-Lite SF was similar to the mock treatment (Figure 6b). Thus, UVA illumination of aged T-Lite SF is necessary for the sensitization of cells to cadmium. The same experiments were performed with Al(OH)3 NMs under UVA and in darkness (Figure 6). Cells pre-exposed to Al-based NMs under UVA (Figure 6c) or in darkness (Figure 6d) were not sensitized to cadmium.

Figure 4. Survival of E. coli MG1655 wild type (wt) and mutants affected in superoxide dismutase ((sodAsodB) and catalase activity (katEkatG) after exposition to TiO2-nanocomposite (TiO2−NC), Al(OH)3 and pristine TiO2 nanomaterials (NMs) in ultra pure water (UPW). Cells in exponential phase (106 cell/mL) were incubated with the NMS for 15 h at 37 °C under UVA 365 nm, 0.365 mW/cm2. From foreground to background [Ti] = 0, 0.7, 7.0, and 70 mg/L or [Al] = 0. 0.05, 0.5, and 4.5 mg/L. Asterisks (*) denote a significant difference between mutants and the WT at the 95% confidence level.

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DISCUSSION Large amounts of nano-TiO2 are produced worldwide and are potentially disseminated in the environment.34 Nano-TiO2 is released by leaching from nanoenabled materials, during their use, such as runoff from facade paints,35 release from textiles,36 use of cosmetics37 and biological human excretion: dietary intake of nano-TiO2 is estimated to be 2.5 mg/individual/d.38 From simulations, 36−56 tons per year of nano-TiO2 from sun creams could be released in coral reef areas39 and 8 kg/y in the lake of Zurich.37 Nano-TiO2 used in sunscreens is made from rutile TiO2 coated with an Al(OH)3 layer and organic polymers to prevent the release of reactive oxygen species, due to photocatalytic activity. After their release in water, the nanocomposites interact with the environment and are modified. The integrity of the Al(OH)3 is compromised by interaction of TiO2−NC used in sunscreen with chlorine from swimming pool water.40 Interaction of T-Lite SF, a TiO2-based nanocomposite with water and UV light contribute to expose the Al(OH)3 layer, with the oxidation and partial release of the polydimethylsiloxane layer.10,11 We have assessed the impact of aged T-Lite SF used in sunscreen on E. coli in pure water, KCl and a buffer medium

Figure 5. Scanning electron microscopy of E. coli interacting with NMs after 2 h of illumination under long wave UV in UPW. Scale bar is 1 μm. (a) E. coli cells alone; (b) E. coli cells and pristine TiO2 rutile [Ti] 7 mg/L (c) and (d) T-Lite SF (TiO2 core coated with Al(OH)3 layer) and E. coli cells.

In UPW, T-Lite SF formed large agglomerates that stuck unevenly on cell surface, some cells being entirely free of particles. Conversely, pristine n-TiO2 agglomerates were smaller and showed a more homogeneous distribution on cell surface than T-Lite SF. Interactions of NMs with cells are related to their zeta potentials.29 Pristine n-TiO2 showed positive zeta potential (26.4 ± 2.2 mV) that favors attractive electrostatic interactions with negatively charged cell surface. TLite SF showed low and near-zero potential, closer to that of nAl(OH)3, which explains the aggregation of particles to each other (homoaggregation) and lower interactions with cell surface. ROS Produced by TiO2 Nanocomposite Induce Neither Lipid Peroxidation nor Mutagenesis. We looked further to some impact of aged T-Lite SF on cell by examining lipid peroxidation and mutagenesis that are a main consequence of 1O2-induced DNA damage.30 DNA damages may result from 5249



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irradiation with visible light revealed a signal, which could be attributed to singlet oxygen formation. Singlet oxygen generation was successful in rutile nano-TiO2-based sunscreen emulsions in DMSO, but was not detected on the amphiphilic TiO2−NC alone.41 To our knowledge, this is the first report of 1 O2 formation from rutile TiO2-based NC under UV exposure. Under higher UVA intensity exposure, aged T-Lite SF generated hydroxyl radicals. Hydroxyl radical production from nano-TiO2 used in sunscreen is already known. Nano-TiO2 extracted from sun creams oxidizes organic molecules and causes DNA damage in human cells.42 Sunscreens containing nano-TiO2, which were inadvertently splashed on durable surface coating, had deleterious effects due to photocatalytic activity.43 In both studies, these injurious effects were due to hydroxyl radicals. We therefore conclude that coating of the TiO2 rutile core with an Al(OH)3 layer in TiO2−NC and embedding in Sibased polymers is not fully effective to preclude the formation of ROS, such as singlet oxygen even under low intensity long wave UV and hydroxyl radicals. From the literature, photocatalytic activity of nano-TiO2 relies on electron transfer, that leads to hydroxyl radical and superoxide radical, and energy transfer responsible for singlet oxygen.44 Modification of the titanium dioxide surface favors the energy transfer pathway and tends to suppress the interfacial electron transfer, without complete inhibition of the latter.44 Upon electron transfer, singlet oxygen results from dismutation of superoxide anion to form hydrogen peroxide and singlet oxygen45 and from Fenton-like reaction between hydrogen peroxide and superoxide to yield singlet oxygen, hydroxyl radical and hydroxyl anion46 The energy transfer mechanism seems possible under low UVA intensity, as we evidenced neither the formation of H2O2 nor the formation of superoxide using probes (APF and XTT), and mutants affected in superoxide dimutase and catalases were not affected in their survival by the interactions with T-Lite SF (Figure 4). The lifetime of 1O2 in biological systems drops from 3 μs to around 100 ns (Moan and Berg, 1991; Schweitzer and Schmidt, 2003) because of the rapid reaction of 1O2 with surrounding biomolecules. However, the production of 1O2 increases when the producer is solubilized in membranes. Singlet oxygen diffuses rapidly and can spend 70% of its lifetime in the membrane of lipidic vesicles.47 Hence, even if the production of 1 O2 can be reduced in a complex buffer, such as MDM, the vicinity of nanomaterials with membranes could increase the effect of singlet oxygen on bacterial cells. The cellular responses to singlet oxygen have only recently begun to be analyzed in contrast to the responses to the classical ROS such as superoxide and peroxides.48 Singlet oxygen is a selective factor affecting bacterial species dynamics in aquatic ecosystems. Some bacterial groups can adapt to 1O2 but others are sensitive.49 1O2 is a signal for bacteria that induces soxRS and oxyR regulons, increases catalase and SODspecific activities and leads to the production of enzymes that protect the cell from oxidative stress. It is known that bacterial adaptive or stress responses to H2O2 and O2•− following low doses protects cells from subsequent exposure to higher doses that would otherwise have been lethal.48 Exposure of bacteria to multiple stresses, such as reactive oxygen species, membrane damage, impacts innate susceptibility of bacteria to a variety of antimicrobials. Initiation of stress responses that positively impact recruitment of resistance determinants or promote physiological changes compromises antimicrobial activity.50

Figure 6. Survival of E. coli in the presence of cadmium after contact with NMs under long wave UV (left panel) or in darkness (right panel). E. coli cells were incubated at 37 °C in UPW, in the presence (gray diamonds) or in the absence (open circles) of nanomaterials under long wave UV illumination (365 nm, 0.365 mW/cm−2) or in darkness and then incubated in darkness with a range of cadmium nitrate concentrations. Upper panel: preincubation of cells and aged TLite SF ([Ti = 7 mg/L, [Al] = 0.5 mg/L) (a) under long wave UV; (b) in darkness. Lower panel: preincubation of n-Al(OH)3 ([Al] = 0.5 mg/L) (c) under long wave UV, [Al] = 0.5 mg/L; (d) in darkness.

containing phosphate and citrate. Two intensities of UVA were used to mimic realistic exposures under winter and summer conditions under long-wave UV (365 nm, 0.365, and 5 mW/ cm2). The effect of T-Lite SF was studied at a Ti concentration of 0, 0.77, 7.7, and 77 mg/L, which is far from environmental concentrations. Concentrations of nanoTiO2 in rivers are currently estimated in the range of 3 ng/L to 1.6 μg/L (Gottschalk et al., 2013) and 4.8 μg/kg in soil (Gottschalk et al., 2009). However, wastewater effluents and biosolid can concentrate 5−15 μg/L of Ti and 1 to 6 μg Ti/mg respectively (Kiser et al., 2009). Still, the production of nanoTiO2 has increased from 3000 tons in 2002 to 44 400 tons in 2009 (Robichaud et al., 2009) and could reach 260,000 tons by 2015 (Robichaud et al., 2009). Due to the systemic escalation of nanoenabled products, the environmental concentrations of TiO2 nanoparticles in soil, water and air are likely to increase in the future. Using fluorescence spectroscopy and EPR, we show that aged T-Lite SF produced 1O2 in pure water and KCl under low UVA intensity. According to Lipovsky,23 singlet oxygen does not form in water suspension of rutile TiO2 under UV light, but 5250



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Nonetheless, the production of 1O2 by aged T-Lite SF under UVA had no impact on the survival of E. coli and mutants, on lipid peroxidation and DNA mutagenesis. Under high UVA intensity, although T-Lite SF produced both HO• and 1O2, the viability was only slightly decreased (Supporting Information Figure S3). We used drastic conditions for exposure of bacteria to nanomaterials (i.e., pure water). In this medium, nanomaterials and cells are likely to strongly interact due to surface charges29 and the absence of organic matter. However, in an environmental medium, the concentration of NMs will be lower. This will disadvantage the homoaggregation of NMs and may be favor the heteroaggregation and interaction with bacteria. Nonetheless, based on these results, as far as the protective Al(OH)3 coating remains intact, aged T-Lite SF nanocomposite appears to be environmentally friendly for bacteria as opposed to pristine nano-TiO2. Though, environmental impact on bacteria goes beyond life or dead. Cells are subjected to various stresses, such as physical stress (UV, temperature...), fluctuations of nutrients, and the presence of compounds belonging to the class of environmental pollutants. Cadmium is a priority contaminant in soil and water. The main source of cadmium contamination results from the use of fertilizers, and spreading of sewage sludge.51 Pre- exposure of cells to aged T-Lite SF and long wave UV sensitized cells to further exposure to cadmium, even after the UV stress had been stopped. Hartman, et al.52 have observed combined toxic effect of pristine TiO2 nanoparticles (2 mg/L) and cadmium on the freshwater green alga Pseudokirchneriella subcapitata. The authors suggest an increase in cadmium uptake due to cell membrane damage caused by TiO2 or a consequence of the sorption of cadmium to the TiO2 NPs. In this study, cells pre-exposed to nonphotocatalytic Al-based NMs under UVA or to T-Lite SF in darkness were not sensitized to cadmium. Therefore, the sensitization did not result from a nano effect, such as sticking of particles and damage to membranes as observed for nano-TiO 2 to Nitrosomonas europae53 or from a Trojan horse effect, the NMs being a support for cadmium. We conclude that sensitization of cells is related to the combination of photocatalytic NMs such as T-Lite SF, and long wave UV, hence to the production of ROS, such as 1O2. The effects of dilute and cocktail pollutions are a major environmental issue. As example, the release of complex mixtures of air pollutants (benzene, heavy metals and polycyclic aromatic hydrocarbons) increases DNA damage in the nasal epithelium of children residing near the power plant as compared to rural controls.54 In soil, unmonitored use of antibiotics and metals from manure causes the emergence and release of antibiotic resistance genes to the environment.55 Kim et al.56 showed that TiO2 rutile nanoparticles in the 40−300 nm range were characterized in sewage sludge solids from wastewater treatment plants. These NMs could adsorb Ag when the sludge were amended with silver nanoparticles, which raises the concern of nanoparticles and metals interaction in the environment. Our results show that TiO2 nanocomposites used in sunscreens, though designed to reduce the formation of ROS, produced ROS as singlet oxygen and hydroxyl radicals under long wave UV after aging of the nanomaterials. Aged T-Lite SF showed no direct effects on cell viability and oxidation of lipids and DNA. However, singlet oxygen production by aged T-Lite SF strongly sensitized E. coli cells to further exposure to cadmium in the absence of UV. The true intention of this study

is not to alarm on direct toxic effects of TiO2-based sunscreens. Nevertheless, our results point out the importance of considering engineered nanomaterials after their transformations-aging and in the context of coeffects with environmental pollutants in order to forecast the risk of exposure to NMs for the environment.

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ASSOCIATED CONTENT

S Supporting Information *

Information includes the physicochemical characterization (Table S1) and transmission electron microscopy images of NMs (Figure S1) used in this study, the results of the lipid peroxidation in E. coli exposed to nanomaterials (NMs) under long wave UV (Figure S2) and the survival of cells under high UVA in the presence of aged T-Lite SF (Figure S3). This material is available free of charge via the Internet at http:// pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 033 442 257 713; fax: 033 442 256 648; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Agence Nationale de la Recherche for funding the AgingNano&Troph project (ANR-08-CESA-001). REFERENCES

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Aged TiO2-Based Nanocomposite Used in Sunscreens Produces

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