Nano-titanium dioxide modulates the dermal sensitization potency of DNCB
- Salik Hussain†1, 2, 4,
- Stijn Smulders†2,
- Vanessa De Vooght2,
- Bert Ectors2,
- Sonja Boland1,
- Francelyne Marano1,
- Kirsten L Van Landuyt3,
- Benoit Nemery2,
- Peter HM Hoet2 and
- Jeroen AJ Vanoirbeek2Email author
© Hussain et al.; licensee BioMed Central Ltd. 2012
Received: 14 October 2011
Accepted: 2 May 2012
Published: 23 May 2012
We determined the ability of a model nanoparticle (NP) (titanium dioxide, TiO2) to modulate sensitization induced by a known potent dermal sensitizer (dinitrochlorobenzene) using a variant of the local lymph node assay called lymph node proliferation assay.
BALB/c mice received sub-cutaneous injections of vehicle (2.5 mM sodium citrate), TiO2 NPs (0.004, 0.04 or 0.4 mg/ml) or pigment particles (0.04 mg/ml) both stabilized in sodium citrate buffer at the base of each ear (2x50μl), before receiving dermal applications (on both ears) of 2,4-Dinitrochlorobenzene (DNCB) (2x25μl of 0.1%) or its vehicle (acetone olive oil – AOO (4:1)) on days 0, 1 and 2. On day 5, the stimulation index (SI) was calculated as a ratio of 3HTdR incorporation in lymphocytes from DNBC-treated mice and AOO-treated controls. In a second experiment the EC3-value for DNCB (0 to 0.1%) was assessed in the absence or presence of 0.04 mg/ml TiO2. In a third experiment, the lymphocyte subpopulations and the cytokine secretion profile were analyzed after TiO2 (0.04 mg/ml) and DNCB (0.1%) treatment.
Injection of NPs in AOO-treated control mice did not have any effect on lymph node (LN) proliferation. DNCB sensitization resulted in LN proliferation, which was further increased by injection of TiO2 NPs before DNCB sensitization. The EC3 of DNCB, with prior injection of vehicle control was 0.041%, while injection with TiO2 decreased the EC3 of DNCB to 0.015%. TiO2 NPs pre-treatment did not alter the lymphocyte subpopulations, but significantly increased the level of IL-4 and decreased IL-10 production in DNCB treated animals.
In conclusion, our study demonstrates that administration of nano-TiO2 increases the dermal sensitization potency of DNCB, by augmenting a Th2 response, showing the immunomodulatory abilities of NPs.
KeywordsNanoparticle Titanium dioxide (TiO2) Lymph node proliferation assay (LNPA) DNCB Skin sensitization
Nanoparticles of titanium dioxide (TiO2) are one of the most abundantly produced and widely utilized nanomaterials , with applications in sunscreens, cosmetics, tooth pastes, and food products [2–4]. Other important industrial applications include water clean-up technology, oxygen sensor and antimicrobial coatings and ceramics. Titanium nanomaterials have proved their potentials in the fields of drug and gene delivery . Although considered as an inert material, titanium alloys and implants have been shown to release both micrometric and nanometric particles and debris in the surrounding body fluids and tissues which can cause health effects either at the implant site or in distant organs [5–8]. A variety of newly developed house hold products (including self-cleaning spray and paint) have been reported to contain TiO2NPs. According to the Project on Emerging Nanotechnologies (Woodrow Wilson International Center for Scholars), as of 10th of March 2011, 1317 consumer products containing NPs are already on the market and if the same trend persists this number is expected to reach 3400 by the year 2020. TiO2 is the 3rd largest material used in the consumer products.
Effects of NPs on biological systems are unknown or under debate [9, 10]. Nevertheless, the widespread uses of TiO2 NPs confirm the possibilities of exposure through ingestion, inhalation and dermal routes. Taken together with the substantial increase of products (in particular skin care products) containing TiO2 nanomaterials, there is an urgent need for assessing these newly developed materials for their possible skin sensitizing potentials as well as for their impact on the skin sensitization caused by the other chemicals.
Allergic skin sensitization caused by chemicals is a serious environmental and occupational health hazard. It is the most frequent manifestation of immunotoxicity in humans . Literature reports identify more than 3700 chemical as skin sensitizers . Based on positive skin sensitization tests in animals, it is predicted that in the near future an increase in the number of chemicals capable of causing contact dermatitis in humans will follow.
Dinitrochlorobenzene (DNCB) is a well-known skin sensitizer. It is the most used chemical to study contact hypersensitivity of the skin and the pathogenesis of contact dermatitis [13, 14]. Contact dermatitis observed after DNCB application is a T-cell mediated immune response . DNCB formed covalent complexes with various proteins after topical application and act as immunogen which are taken up by antigen presenting cells (APC), processed and presented to T cells for activation.
The Local Lymph Node Assay (LLNA) is an OECD approved protocol to assess dermal sensitizing capacity of chemicals (OECD guideline no. 429, 2002) . A modified version of this test, lymph node proliferation assay (LNPA), has recently been suggested as more appropriate test methodology for the evaluation of NP induced delayed type hypersensitivity reaction [17, 18]. The major advantage of LNPA/LLNA includes the possibility to calculate chemicals relative potency of inducing dermal sensitization.
The aim of this study was to evaluate the effect of a prior administration of TiO2 NPs on the dermal sensitization potential of DNCB. We hypothesized that TiO2 NPs might act as an adjuvant for a skin sensitizer, such as DNCB. To our knowledge, this is the first study to describe the effect of NPs on the dermal sensitization potential of a well-known chemical sensitizer.
Lymph node cell subpopulations (x10 6 ) per lymph node
0.66 ± 0.21
2.06 ± 0.56***
1.16 ± 0.57
2.27 ± 0.59+++
CD3 + CD4 +
0.47 ± 0.15
1.47 ± 0.41***
0.81 ± 0.38
1.56 ± 0.43+++
CD3 + CD4 + CD25 +
0.04 ± 0.01
0.16 ± 0.05***
0.06 ± 0.03
0.13 ± 0.02++
CD3 + CD8 +
0.18 ± 0.06
0.59 ± 0.15***
0.34 ± 0.20
0.68 ± 0.19+++
0.12 ± 0.04
0.62 ± 0.22***
0.23 ± 0.13
0.80 ± 0.30+++
Our objective was to evaluate the effects of non-biodegradable/biopersistant solid TiO2 NPs on the classical hypersensitivity reaction to a well-known potent dermal sensitizer (DNCB). To the best of our knowledge, there is no information available on the effect of manufactured NPs on the dermal sensitization potential of chemicals, in general, and of DNCB in particular. Recently, sensitizing potentials of biodegradable particles (ethosomes) was described, but such information about environmentally relevant solid NPs is lacking [19–21]. Here we demonstrate that TiO2 NPs (0.04 mg/ml) act as an immune-stimulator on the dermal sensitization capacity of DNCB. The stimulation of the dermal sensitization was coupled with a significant change in cytokine release which corresponds with the induction of a Th2 response. The experiments confirmed our hypothesis that pre-treatment with TiO2 NPs modulate sensitization to DNCB.
There is conflicting evidence about the skin penetration of nanomaterials [22, 23]. However, it has been suggested that NPs could pass through the stratum corneum of the skin using intercellular channels or hair follicles and penetrate into deeper skin layers [24, 25]. A thorough search of the available literature indicates that stratum corneum is an effective barrier against the uptake of TiO2 NPs in healthy skin. However, various research publications anticipate the possibility of penetration of TiO2 NPs to the deeper layers of skin/viable skin in case of local damage (sun burns etc) or when lipid vesicles formulations are involved [19, 20, 26]. For example, a sub-erythemal dose ultraviolet radiation exposure (UVB 270 mJ/cm2) in mice has been shown to allow the penetration of 45 nm quantum dot NPs to deep dermis . In another study, it has been observed that 7 nm TiO2 NP exposure with UV irradiation can lead to increased skin barrier dysfunction and possible aggravation of contact dermatitis due to increased invasion of Staphylococcus aureus . Interestingly, people tend to apply more sun screens containing NPs in case they have damaged skin (sunburns, burns) when the barrier function of skin is already impaired. It has also been reported that in a skin barrier dysfunction conditions (mite allergen exposure) intradermal exposure to rutile TiO2 NPs can lead to aggravation of the atopic skin lesions . Particles which penetrate normal or damaged skin are taken up by antigen presenting cells (APC), such as Langerhans cells or dendritic cells, and subsequently removed via the lymphatic system [9, 30]. In view of these considerations, along with the possibility of exposure in individuals with pathological lesions of the skin, subcutaneous route becomes a relevant route to study NP induced effects on dermal sensitization. TiO2 NP concentrations used in this study are based on literature reports by us and others describing these as non-cytotoxic concentrations in vitro and in vivo [31, 32]. Moreover, we did not observe any type of local injury around the site of injection of NPs.
Our results indicate that TiO2 NPs do not show dermal sensitization potential after a single subcutaneous injection, which is in accordance with findings in literature . However, subcutaneous presence of TiO2 NPs significantly increases the skin sensitization potential of DNCB, which is not the case with subcutaneous presence of pigment TiO2 particles before dermal sensitization. It is possible that increased sensitization after NP exposures is due to binding of the antigen with the NPs, leading to formation of depot of antigen which is better recognized by skin APCs. It has been proposed that ultrafine TiO2 particles (below 30 nm) bind more ovalbumin per mass unit than fine particles and this binding may lead to a depot of antigen leading to increased antigenicity . Moreover, it is a well accepted fact that professional APCs are more readily stimulated by the particulate antigens, thus adsorption of antigen per se can increase the antigenicity . Other possible explanation might be physical interactions between the NPs and APCs. Impurities cannot be a confounding factor in our experiments as there were no detectable amounts present in TiO2 NPs utilized in this study.
The results of the present study demonstrate that pre-exposure to TiO2 NPs does not interfere with the immune system if followed by a sham dermal treatment (TiO2/Veh). However, when pre-exposure to TiO2 NPs is followed by DNCB sensitization, a Th2 favoured immune response in regional lymph node cells develops, with increased IL-4 and decreased IL-10 levels, while DNCB itself is a known potent Th1 responder . The apparent IFN-γ secretion confirms the DNCB-induced Th1 response, even with prior injection of TiO2 NPs. Nevertheless, TiO2 NPs injection followed by DNCB sensitization results in significantly increased levels of IL-4, demonstrating Th2 stimulation. In addition, we found a decrease in IL-10 secretion. IL-10 is a cytokine released by several cell types, such as monocytes, activated T cells, Th2 cells, mast cells and regulatory T cells. IL-10 is capable of inhibiting pro-inflammatory responses and is suggested to play a major role in maintenance of self-tolerance . We think that sub-cutaneous exposure to TiO2 in DNCB sensitized mice, decreased IL-10, and thereby allowing the development of a Th2 response, independent of the presence of the Th1 response (levels of IFN-γ are maintained).
Allergic sensitization reactions are the first step against the “foreign” materials, and are either Th1 or Th2 polarized. It has been shown that particles themselves can act as modulating agents in skewing the Th responses. Impact of particles on the skewing of Th response is largely dependent on the chemical nature and characteristics of the materials. Larsen et al, found that TiO2 NPs promote allergic sensitization to ovalbumin (IgE and IgG1 levels) and thus primes a Th2 dominated immune response . Diesel exhaust can promote both Th1 and Th2 responses [37, 38]. Carbon nanotubes either amplify Th1 (MWCNT) or Th2 (SWCNT) or both (MWCNT) responses [39, 40]. These studies are done in the models of respiratory allergy using ovalbumin as sensitizing agent. However, respiratory allergic responses have already been shown to be more prone to Th2 skewing while skin sensitization responses are mostly Th1 dependent [41, 42]. It is interesting to note that although we observe a skewing of immune response towards Th2 we still observe a shift in the potency of DNCB, with almost a 3-fold fold decrease in EC3.
In conclusion, we have demonstrated that TiO2 NPs are effective in modulating the chemical-induced in vivo dermal sensitization. They probably act as adjuvant to increase the dermal sensitization capacity of a model chemical skin sensitizer (DNCB). These findings will be helpful in understanding the NPs induced health effects and will help in understanding the interactions of NPs with other sensitizing agents.
2,4-Dinitrochorobenzene was purchased from Sigma-Aldrich (Bornem, Belgium). Trichloroacetic acid (TCA) and acetone were obtained from Sigma-Aldrich (Bornem, Belgium). Pentobarbital (Nembutal) was obtained from Sanofi Santé Animale (CEVA, Brussels, Belgium). Acetone-olive oil vehicle (AOO) used to dissolve DNCB was composed of a mixture of 4 volumes of acetone and 1 volume of olive oil (Selection de Almazara, Carbonell, Madrid, Spain). DNCB concentration is given as percent (w/v) in AOO. Hanks Balanced Salt solution (HBSS) was purchased from Invitrogen (Merelbeke, Belgium) and [Methyl-3 H]-thymidine (3HTdR) was purchased from ICN Pharmaceuticals (Asse, Belgium).
TiO2 NPs (99.9% anatase) of 10–25 nm diameter (15 nm average diameter) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). These particles were prepared by sol–gel process and were used without any post production surface treatments/modifications. Pigment TiO2 was purchased from Cinkarna (Celje, Slovenia). Freshly prepared NPs suspensions at desired concentrations in 2.5 mM sodium Citrate were utilized to treat the mice.
Physico-chemical Characterization of NPs
Dynamic Light Scattering
DLS analysis of TiO2 pigment and NPs was performed according to the protocol described by us previously . Briefly TiO2 pigment and NPs were diluted in 2.5 mM sodium citrate (pH 6.95, ionic strength (I)515 mM), ultrasonicated and analyzed using Brookhaven 90 Plus instrument (scattering angle: 90°, wavelength: 659 nm, power 15 mW). Correlation functions were analyzed with the Clementine package (maximum entropy method) for Igor Pro 6.02A. This resulted in intensity weighted distribution functions versus decay times. By converting the decay times with instrument parameters and physical parameters to hydrodynamic diameters, an intensity weighted size distribution was obtained. A lognormal fit was applied on each population resulting in the average hydrodynamic diameter of the population.
Zeta Potential (ζ ) Measurements
Detailed protocol of ζ potential measurement is published elsewhere  was measured with a Brookhaven 90Plus/ZetaPlus instrument applying electrophoretic light scattering. A primary and reference beam (659 nm, 35 mW), modulated optics and a dip-in electrode system were used. The frequency shift of scattered light (relative to the reference beam) from a charged particle moving in an electric field is related to the electrophoretic mobility of the particle. The Smoluchowski limit was used to calculate the zeta potential from the electrophoretic mobility.
Transmission electron microscopy (TEM)
Suspensions of the TiO2 particles were applied on formvar-coated cupper mesh grids. After drying overnight, the NPs were characterized by transmission electron microscopy (TEM) (JEOL JEM-1200 EX-II, Tokyo, Japan) at a magnification of 20.000-200.000 x.
Male BALB/c mice (approximately 20 g, 6 weeks old) were obtained from Harlan (Horst, The Netherlands). The mice were housed in a conventional animal house with 12-hour dark/light cycles. They were housed in plastic cages with filter tops and received lightly acidified water and pelleted food (Trouw Nutrition, Gent, Belgium) ad libitum. All experimental procedures were approved by the local Ethical Committee for Animal Experiments.
Lymph Node Proliferation Assay (LNPA)
In a first experiment, mice were subcutaneously injected with 2x50μl of a 0.004 (low dose), 0.04 (medium dose) or 0.4 (high dose) mg/ml NP suspension, 0.04 (medium dose) mg/ml pigment particles or vehicle on the area medial to the implantation of ear lobes. One hour later, the mice were dermally treated on both ears with (25 μl) of 0.1% DNCB in AOO, or AOO alone and this for 3 consecutive days. On day 5, the LNPA was performed and the SI calculated.
In a second experiment, mice were subcutaneously injected with 2x50μl of 0.04 (medium dose) mg/ml NP suspension or vehicle on the area medial to the implantation of ear lobes. Afterwards, the mice were treated on each ear with DNCB (0.025, 0.050 or 0.1%) in AOO, or AOO alone, for 3 consecutive days. On day 5, the LNPA was performed, the SI calculated, from which the EC3 (effective concentration yielding a Si of 3) was determined.
Lymph node cell analysis
Cell subpopulation analysis:
Cells were seeded into 48-well culture plates at a density of 106 cells/ml and incubated in complete RPMI-1640 medium for 42 h containing 2.5 μg/ml concanavaline A (ConA) (Sigma–Aldrich, Bornem, Belgium). Culture supernatant was collected, centrifuged (1000 g, 15 minutes at 4°C) and stored at −80°C till further analysis. Levels of IL-2, IL-4, IL-10, IL-13, IL-17 and interferon gamma (IFN-γ) were measured via Cytometric Bead Array and analyzed with the FCAP Array Software on the FACSArray (BD Biosciences, Erembodegem, Belgium). Lower detection limits were 0.2 pg/ml, 0.3 pg/ml, 9.6 pg/ml, 2.4 pg/ml, 0.95 pg/ml and 0.5 pg/ml, respectively.
Data are presented as means and SD. All the data were log transformed for statistical analysis (Graphpad Prism 5.01, Graphpad Software, Inc, San Diego, CA). Data of Table 1, Figures 2 and 4 were analysed by a one-way ANOVA followed by Bonferroni post hoc test. In the post-hoc test, four different comparisons were performed: Veh/AOO vs. Veh/DNCB, Veh/AOO vs. TiO2/AOO, TiO2/AOO vs. TiO2/DNCB and Veh/DNCB vs. TiO2/DNCB. Figure 3 was analysed using a two-way ANOVA followed by a bonferroni post hoc test, comparing Veh vs TiO2 injection prior to DNCB sensitization. A level of p < 0.05 (two tailed) was considered as significant.
This work was supported by ANR grant n° 05 9 9–05 SET 024–01, ANR grant n° 06 SEST 24–01, CAMPLP, Legs Poix, the Interuniversity Attraction Pole Program, Belgian State, Belgian Science Policy P6/35, from the ‘Fonds voor Wetenschappelijk Onderzoek Vlaanderen’ (FWO), FWO G.0547.08 and the EU-FP7 project “Nanohouse” Grant Agreement n°247810. J.V. and V.D are a post-doctoral fellow of the FWO.
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