Journal of Nanomaterials & Molecular NanotechnologyISSN: 2324-8777

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Research Article, J Nanomater Mol Nanotechnol S Vol: 0 Issue: 2

Effects of TiO2 Nanoparticles on Human Myelomonocytic Cell Line THP-1

Kathrin Becker1, Sebastian Schroecksnadel1, Simon Geisler1, Marie Carriere2, Johanna M. Gostner3, Florian Überall3,Nathalie Herlin4 and Dietmar Fuchs1*
1Division of Biological Chemistry, Biocenter, Innsbruck Medical University,Innsbruck, Austria
2Laboratoire Lesion des Acides Nucleiques, CEA Grenoble, Grenoble, France
3Division of Medical Biochemistry, Biocenter, Innsbruck Medical University,Innsbruck, Austria
4CEA IRAMIS NIMBE LEDNA, Laboratoire Francis Perrin CEA-CNRS URA 2453, Bat 522 CEA Saclay, 91191 Gif/Yvette Cedex, France
Corresponding author : Dr. Dietmar Fuchs
Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innrain 80, Innsbruck, Austria
Tel: +43 512 9003 70350;Fax: +43 512 9003 70350
Received: May 10, 2014 Accepted: August 29, 2014 Published: September 03, 2014
Citation: Becker K, Schroecksnadel S, Geisler S, Carriere M, Gostner JM et al., (2014) Effects of TiO2 Nanoparticles on Human Myelomonocytic Cell Line THP-1. J Nanomater Mol Nanotechnol S2:005. doi:10.4172/2324-8777.S2-005


Effects of TiO2 Nanoparticles on Human Myelomonocytic Cell Line THP-1

The frequency of exposure to nanoparticles has remarkably increased throughout recent years. Titanium dioxide (TiO2) materials are commonly used for consumer and health care products as well as for technical and medicinal applications. Recently, both suppressive and stimulatory effects of TiO2 nanoparticles on human peripheral blood mononuclear cells have been shown, indicating T-cell - macrophage interactions to be an important target for nanoparticle action.
To address the impact of nanoparticles on isolated monocytes, we investigated effects of TiO2 nanoparticles on the human myelomonocytic cell line THP-1 in vitro and monitored the influence
of particle treatment on tryptophan breakdown in unstimulated and lipopolysaccharide (LPS)-stimulated cells in this study.

Keywords: Titanium dioxide; Nanoparticles; Immune system; THP-1; Indoleamine 2,3-dioxygenase


Titanium dioxide; Nanoparticles; Immune system; THP-1; Indoleamine 2,3-dioxygenase


The intensive application of nanomaterials has resulted in an enormously increased exposure of humans to these materials [1,2]. Aside from many positive properties of the nanomaterial, which make them desirable for such many applications, they may represent a risk factor for human health. The difference of bulk materials compared to the small sized (1-100nm) nanoparticles is based on the drastic increase of the total surface area together with the physicochemical properties of nanoparticles. These changes in shape, size, surface characteristics and inner structures may lead to the exertion of cell toxicity [3-7]. In our previous study we suggested to use bulk material as an additional control when investigating nanoparticle activities [8].
TiO2 is one of the best studied nanomaterials; it is applied in food, chemical, electrical or electronic products. Interestingly, TiO2 particles are the most commonly used photosensitizer in photodynamic therapy for cancer [9] and they are used as heat generation agent in magnetic hyperthermia therapy [10]. TiO2 exhibits lower toxicological effects than other similar metal oxide nanoparticles, like zink oxide or aluminum oxide as well as ferric oxide [11].
Results from Chen et al. indicate a genotoxic potential of TiO2 nanoparticles that was observed in rats in vivo after intragastric administration as well as in particle treated cell cultures in vitro [12]. However since now, data assessing human TiO2 exposures are rare and most information on the mode of particle action derived from cell and molecular biological in vitro findings or from animal studies. In particular, potential immune modulating properties of nanoparticles are in the focus of research. Due to the immune stimulatory properties of some nanoparticles, they are under investigations regarding their use as drug delivery systems or vaccine formulations [13,14]. Gonçalves et al. found that exposure to TiO2 induced neutrophil influx and production of pro-inflammatory mediators in vivo in a murine inflammation model [15]. Immunotoxicity of nanoparticles may be related to their potential to provoke inflammatory cascades. High interleukin-(IL)-1β release [16,17], toll like receptor (TLR) and nuclear factor kappa B (NF-κB) activation upon nanoparticle exposition [14,18] indicate the activation of immune responses. Induction of expression of inflammatory cytokines and NF-κB activation was observed also in animal studies [19,20]. Of note, TiO2 nanoparticles are able to induce reactive oxygen species (ROS) formation [14,21]. There is increasing evidence that TiO2 particles can cross the blood-brain-barrier thereby inducing pathological changes [22]. Huerta-García et al. demonstrated that exposure to TiO2 in brain cells resulted in an induction of oxidative stress in vitro, which correlated with the activation of antioxidant enzyme expression and lipoperoxidation [23].
In our previous study we could show that TiO2 nanoparticles interfere with T-cell - macrophage interactions by using a model system of mitogen stimulated peripheral blood mononuclear cells (PBMC) to investigate Th1-type immune activation cascades [8]. Upon T helper cell type 1 (Th1) immune activation, the cytokine interferon-γ (IFN-γ) is released from T-cells. IFN-γ represents an important trigger for ROS production in human macrophages as part of their cytocidal and antimicrobial repertoire [24]. Furthermore, IFN-γ induces the enzymes GTP-cyclohydrolase I (GTP-GCH-I) and indoleamine 2,3-dioxygenase (IDO). IDO catalyzes the breakdown of the essential amino acid tryptophan into kynurenine. High IDO activity, which can be estimated by the kynurenine to tryptophan ratio (Kyn/Trp), indicates a strong cellular immune activation. Tryptophan depletion by IDO limits growth of invading pathogens and tumor cells and represents a negative feedback mechanism that regulates immune response [25]. GTP-GCH-I produces the central inflammation marker neopterin [26]. Both neopterin and tryptophan breakdown are considered as biomarkers for Th1-type responses in vitro as well as in vivo [25,27].
In that previous study in human peripheral blood mononuclear cells (PBMC) we observed cellular responses to TiO2 treatments that suggested potential pro-oxidative properties of the particles [8]. IDO activity was decreased in mitogen-activated PBMC upon particle treatment at higher treatment concentrations, indicating immunosuppressive effects. But measured neopterin levels were elevated in both unstimulated and stimulated cells, thus suggesting immune activation. Thus, these nanoparticles might affect monocytes/ macrophages in a different manner than T-cells.
Here, we continued the study to investigate the effect of TiO2 nanoparticles on the myelomonocytic cell line THP-1 and by analyzing potential pro-oxidative properties in PBMC. THP-1 cells have been described previously as a suitable cell system for the screening of immunomodulatory nanomaterials by using tryptophan breakdown rate as indicator [28], in analogy to the PBMC system [27]. In the present study, this stable cellular model was employed to measure the influence of two types of TiO2 nanoparticles (P25 and OCTi60) compared to commercial TiO2 bulk material on the Th1- type immune activation in THP1 cells in vitro. Previously, THP-1 cells were shown to exhibit a relative resistance to TiO2 nanoparticlemediated toxicity [29] and no impact on cell viability was observed in an interlaboratory evaluation study, except for a nanobelt form of TiO2, up to a concentration of 100 μg/mL [17]. Furthermore, previous results indicated pro-oxidative properties of these materials, thus the potential to induce ROS formation was analyzed in PBMC.

Materials and Methods

TiO2 materials
Three different types of TiO2 materials were tested. Two types were from commercial source as TiO2 bulk material, which was purchased from Sigma-Aldrich (≥99% purity, No: 14021, Paris, France) and P25 nanomaterials, which was from Degussa-Evonik (≥99.5% purity, Frankfurt, Germany). The third type of material, OCTi60 nanoparticles, was a laboratory sample, produced at the Service des Photons, Paris, France.
Lipopolysaccharide (LPS) and phytohemagglutinin (PHA) were purchased from Sigma-Aldrich (No: L6529, 61764, Vienna, Austria), dissolved in phosphate buffered saline (PBS) and stored at -20°C until use.
Preparation of TiO2 materials
For the experiments we compared TiO2 materials from commercial source (bulk material and P25 nanoparticles) as well as a laboratory nanoparticle sample (OCTi60). All materials were prepared as published previously [8]. Materials were mixed with sterile water, sonicated, diluted in foetal calf serum (FCS, S0115, Biochrom, Berlin, Germany) and mixed by stirring on a magnetic bar. Further dilutions were made with culture media RPMI 1640 (E15840, PAA, Linz, Austria) containing 10% FCS.
Cell culture and PBMC isolation
THP-1 is a human myelomonocytic cell line derived from a patient suffering from acute monocytic leukemia (No: ACC-16, DMSZ, Braunschweig, Germany). THP-1 cells were cultured in the standard medium RPMI 1640 containing 300 mg/L L-glutamine (E15840, PAA, Linz, Austria), supplemented with 10% heat inactivated fetal bovine serum (No: S0115, FBS, Biochrom, Berlin, Germany), without antibiotics at 37°C in a humidified atmosphere containing 5% CO2.
PBMC were isolated from healthy donors, which confirmed that their donated blood might be used for scientific purposes in case when it was not selected for transfusion. Blood cell separation by density gradient procedure was described earlier [30]. Obtained PBMC were washed three times in phosphate buffered saline containing 1 μmol/L ethylenediaminetetraacetic acid and subsequently cultivated in RPMI 1640 supplemented with L-glutamine, 10% heat-inactivated FCS (No: S0115, FBS, Biochrom, Berlin,Germany) and 50 μg/mL gentamicin (No.:17-519Z, Lonza-BioWhittaker, Walkersville, MD, USA) at 37°C in a humidified atmosphere containing 5% CO2.
Measurements of tryptophan and kynurenine concentrations
THP-1 cells were seeded at a density of 3 x 10^6 cells/3mL/well in RPMI 1640 in a 12-well plate. The different types of TiO2 materials, P25 and OCTi60 and TiO2 bulk material were added in increasing amounts starting from 9.4 – 150 μg/mL. Afterwards cells were either stimulated with 1μg/mL lipopolysaccharide (LPS; No: L6529, Sigma- Aldrich, Vienna, Austria) or left unstimulated and were incubated for 48 h at 37°C. Then, supernatants were harvested by centrifugation and frozen at -20°C until measurements were performed.
Tryptophan and kynurenine concentrations were measured by HPLC (ProStar Varian, Palo Alto, CA, USA) using rp-18 columns (No: 150231, Merck, Darmstadt, Germany) and acetate buffer at a flow-rate of 1 mL/min and 3-nitro-L-tyrosine (No: N7389, Sigma Aldrich, Vienna, Austria) as an internal standard [31,32]. Kynurenine (K8625, Sigma-Aldrich, Vienna, Austria) and 3-nitro-L-tyrosine were detected by UV-absorbance at 360 nm wavelengths (Shimadzu SPD-6A UV detector, Korneuburg, Austria), tryptophan by its native fluorescence (excitation wavelength 286 nm, emission wavelength 366 nm, ProStar 360, Varian, Palo Alto, CA, USA). The kynurenine to tryptophan ratio (Kyn/Trp) was calculated to estimate IDO activity and expressed in μmol Kyn/mmol Trp [31]. All chemicals for HPLC method were obtained from Sigma-Aldrich (Vienna, Austria) and where of highest available purity grade. The sensitivity of the measurements is 0.5 μmol/L kynurenine and 0.1 μmol/L tryptophan.
Cellular antioxidant activity (CAA) assay
Relative changes of intracellular ROS levels in cells can be monitored by using the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA, ≥ 97% purity, D6883, Sigma Aldrich, Vienna, Austria) as a substrate. DCFH-DA diffuses through cell membranes and is hydrolyzed by intracellular esterases to non-fluorescent 2’,7’-dichlorofluorescin (DCFH), which is then trapped within the cell. In the presence of ROS, DCFH is rapidly oxidized to highly fluorescent 2’-7’-dichlorofluorescein (DCF), whose intensity is proportional to the amount of intracellular ROS [33,34]. 1.5x10^5 PBMC/90 μL/well were seeded in hank´s balanced salt solution (HBSS, H8264, Sigma Aldrich, Vienna, Austria) containing 25 μM DCFH-DA in 96-well microplates and incubated for 60 min at 37°C. Subsequently, quadruplicate well were additionally treated with solvent [1.5% H2O] or increasing doses of the nanoparticles [18.8 – 150 μg/mL] for 60 min. Afterwards, solutions were removed after centrifugation at 1250 rpm, cells were washed twice with prewarmed PBS and incubated for additional 45 min at 37°C either with 90 μL HBSS for the assessment of potential nanoparticle induced ROS-formation or HBSS containing 600 μM 2,2’-azobis(2- methylpropionamidine) dihydrochloride (AAPH) (No: 017-11062, Wako Chemicals, Neuss, Germany) for the assessment of effects under oxidative stress conditions. The fluorescence of DCF (485/538 nm) was determined with a Fluoroskan Ascent FL plate reader (Thermo Labsystems, Waltham, Massachusetts, USA).
Kyn/Trp results are expressed as percent of unstimulated and LPS-stimulated control cells. ROS formation is shown as DCF fluorescence (485/538 nm). All results are shown as means of +/- standard error of the mean (S.E.M.). For statistical analysis of the results, SPSS version 21 software was used. P-values <0.05 were considered to indicate statistical significances.


Characterization of the different types of particles
The three different samples differ in average particles diameter and the structural properties. P25 (anatase/rutile 85/15) and OCTi60 (anatase/rutile 90/10) TiO2 nanoparticles sizes were 23 nm diameter for P25 and 10 nm for OCTi60 according to TEM, and 25 nm diameter of P25 (60 m2/g) and 16 nm of OCTi60 (95 m2/g) according to BET. Diameter in water (Z average) was 140 for P25 and 70 for OCTi60 material, Z-potential in water was +1 for P25 and -6 for OCTi60. In medium diameter (Z average) was 220 for P25 and 170 for OCTi60 material, Z-potential in medium was -8 to -12 of P25 and -9 to -12 for OCTi60 nanoparticles.
TiO2 bulk material has a more complex particle size distribution. No detailed distribution of the average diameter could be denoted. Hydrodynamic measurements of P25 and OCTi60 in water showed that the laboratory sample, OCTi60, had a smaller average diameter compared to P25 TiO2. The z-average values in the range of 100 nm indicated the presence of a limited amount of agglomerates in the suspension. The hydrodynamic volume increased in both cases after transfer to the biologic medium with a decrease of the zeta potential, which was -9 mV just after transfer to the medium and still decreased to -12 mV after 48 h. On this time period, no significant change was seen in the signal of the turbidimeter, which indicated good stability of both suspensions. Characterization was performed at Service des Photons, Paris, France as reported previously [8].
Influence of TiO2 materials on tryptophan breakdown
THP-1 cells decreased tryptophan in the medium to about 60% of initial content (~37 μmol/L) during the 48 h of incubation (unstimulated cells: 63.6 ± 1.9%, LPS-stimulated cells: 59.2 ± 4.9%). Kynurenine formation was 3.8 times higher in PHA stimulated than in unstimulated cells after this incubation period, thus also kynurenine to tryptophan ratio increased to comparable extent.
Treatment of THP-1 cells with bulk material (number of biological replicates, n=2) did not influence tryptophan breakdown in unstimulated cells as compared to control cells. However, a trend towards higher kynurenine formation was observable. Kynurenine concentrations raised in a dose-dependent manner, treatment of cells with the highest bulk material concentration (150 μg/mL) led to the formation of 187.7 ± 49.9% kynurenine compared to untreated control. Accordingly, Kyn/Trp ratio was elevated up to 1.3 - 2.1- fold compared to control with highest concentration (150 μg/mL), however not significantly different from results obtained with lower concentrations (Figure 1A).
Figure 1: Immunomodulatory effects of TiO2 nanoparticles. The effects of nanoparticle treatment on indoleamine 2,3-dioxygenase activity is indicated by the kynurenine to tryptophan ratio. White bars indicate unstimulated THP- 1 cells, black bars indicate cells stimulated with 1 μg/mL lipopolysaccharide (LPS). Cells were preincubated with different concentrations of either TiO2 bulk material (A), OCTi60 (B) or P25 (C) particles, before stimulation with LPS or not, respectively. Data shown are mean values of more than two independent experiments run in duplicates. No treatment group showed significantly difference to the respective control group.
Particle treatment with OCTi60 increased kynurenine formation and Kyn/Trp in THP-1 cells only about 1.1-fold (n=4). Increase was similar for the whole treatment concentrations (no dose-dependency observable) (Figure 1B).
Addition of P25 in the same concentration range (9.4 – 150 μg/ mL) did not affect tryptophan concentrations as compared to control cells (n=3). Kynurenine levels as well as the Kyn/Trp were elevated to maximum with the highest treatment concentrations to 166 ± 40.1% (kynurenine formation) and 161 ± 46.5% (Kyn/Trp) (Figure 1C).
Both tryptophan and kynurenine concentrations were not affected with all particle treatment in LPS-stimulated cells. Of note, all the reported changes were not statistically significant, all p-values were higher than 0.5.
Influence of TiO2 nanoparticles on oxidative stress
PBMC were preincubated for 1h with increasing concentrations of 25 nm-sized material P25, OCTi60 or medium substituted with 1.5% water (solvent control) and incubated with an oxidation-sensitive intracellular trapped substrate for 1h. Both materials showed the capacity to significantly induce oxidative stress in a dose-dependent manner (n=3). Although overall fluorescent changes were low as indicated by the absolute fluorescence values (Figure 2A), the highest treatment concentrations of 150 μg/mL led to an increase of 191 ± 6.9% with OCTi60 and 193 ± 17.7% as compared to the untreated control, which was set as 100%.
Figure 2: Interference of nanoparticles with ROS-formation. (A) Human peripheral blood mononuclear cells (PBMC) were incubated with different concentrations of OCTi60 and P25. (B) Effect of nanoparticle-treated PBMC was analyzed under elevated oxidative stress conditions. Mean values ± SEM of three independent experiments are shown. (*p ≤ 0.5 compared to control (ctrl)).
Upon induction of oxidative stress conditions by adding a peroxyl-radical forming agent, absolute fluorescence values increased significantly (Figure 2B). Under these conditions, influence of particles was less strong and could superinduce ROS-formation only at higher treatment concentrations. With the highest treatment dose, materials increased fluorescence to 113.8 ± 5.4% with OCTi60 and 114.8 ± 7.6% as compared to the untreated control.


The aim of this study was to (i) investigate the effect of TiO2 materials, which we previously investigated in PBMC [8], on the myelomonocytic cell line THP-1 and to (ii) further elucidate the potential pro-oxidative properties of these materials.
Investigating the effect of the particles on central immunobiochemical pathways such as tryptophan breakdown via IDO does not only uncover potential immunomodulatory properties of such new materials, but by using different cell systems, also celltype specific responses can be validated and immunotoxicologial effect can be analyzed in more detail.
When comparing the data of this follow-up study using myelomonocytic THP-1 cells with the PBMC data, differences in cell responses can be observed. A significant suppressive effect on IDO activity in mitogen-stimulated PBMC was observed for treatment with bulk material and OCTi60 and a similar but less strong trend was found for P25 [8]. Interestingly, observed effects showed a biphasic distribution. Low-concentration effect suggested immune-activating properties, e.g., as IDO activity was significantly enhanced upon low-dose P25 treatment. Additionally, the oxidative stress and inflammation marker neopterin, which is produced by GCH in human macrophages, was dose-dependently increased with all treatments in unstimulated PBMC and with OCTi60 and bulk material treatment in mitogen-stimulated cells. Regarding the strength of the immunomodulatory properties, OCTi60 and bulk material exerted stronger effects than P25 in PBMC.
In the current study, the effect of nanoparticles on ROS-formation in PBMC was assessed using a fluorescent-based detection method (CAA-assay). Both nanoparticle materials were demonstrated to exert pro-oxidative action. Effects were preferentially detected in the unstimulated PBMC but less when cells were additionally exposed to AAPH which generates peroxyl radicals. Obviously, the effects, which could be induced by TiO2 materials, were much less strong than the effect achieved with the radical regenerator AAPH. In stimulated cells, particle-dependent superinduction of ROS might be a plausible scenario. Although probably this effect will be minor in comparison to other ROS triggering events, it could support sustained activation and counteract a successful resolution of inflammation.
TiO2-induced pro-oxidative effects might trigger immune activation. This suggestion is in line with the observation of the effects of TiO2 materials on the Kyn/Trp in THP-1 cells. Moreover, the increase of neopterin production found earlier in unstimulated PBMC [8] pointed towards an activation of pro-inflammatory cascades. Interestingly, all materials showed effects on IDO activity, indicated by Kyn/Trp, in unstimulated but not stimulated THP-1 cells in this study (bulk > P25 > OCTi60), although effects were not of significance due to larger interassay variations. The standard deviations with this assay appear rather high, but potential interferences of the particle material with the analytical equipment before and during the study were carefully addressed. We hypothesize, that interassay variations are influenced by the aggregation and sedimentation behavior of the particles in the stock solution and in the diluted treatment solutions. This aspect is especially valid for the experiment with bulk material.
The particle size may not be solely responsible for the immunobiological effects of TiO2 materials. Bulk material has almost similar effects than P25 (25nm diameter) and the smallest OCTi60 with 10nm diameter had the lowest influence on tryptophan breakdown in vitro. Nevertheless, a reason for the different bioactivity could be the differing amount of anatase and rutile in the composition of the particles. OCTi60, with a somewhat smaller amount of rutile but more anatase compared to P25 (anatase to rutile ratio for OCTi60: 90/10 and for P25: 85/15), had smaller influence on KYN/TRP levels than P25. Of note, the presence of nano-sized particles also in the bulk material cannot be excluded.
Monocyte-derived macrophages are important targets of nanoparticles. In animal experiments, ultrafine TiO2 particles were shown to induce toxicity in rat alveolar macrophages due to prooxidant mechanism as well as antioxidant depletion [35]. Numano et al. reported that particle treatment in rats resulted in an increased number of alveolar macrophages in the lungs, as well as increased levels of 8-hydroxydeoxyguanosine, a marker of oxidative stressderived DNA damage, and macrophage inflammatory protein 1 (MIP1) [36].
Thus, detailed knowledge of immunobiochemical changes induced by nanoparticles in different cell types is necessary to better understand the complex immunomodulatory properties of such materials and will help to evaluate potential immunotoxicity.


Support by the Austrian Research Funds (project 25150-B13) and the European Commission (ERA NET–New INDIGO Program, NanoLINEN is gratefully acknowledged.


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