Andreea R Lupu1,2#*, Lidia Cremer2 and Traian Popescu3#
1Immunobiology Laboratory, "Victor Babes" National Institute of Pathology, Bucharest, Romania
2Immunomodulation Group, "Cantacuzino" National Institute for Research/Development in Microbiology and Immunology, Bucharest, Romania
3LASDAM Laboratory, National Institute of Materials Physics, Bucharest, Romania
#These authors contributed equally to this work
Received: 12 March, 2015; Accepted: 11 April, 2015; Published: 13 April, 2015
Andreea-Roxana Lupu, Immunobiology Laboratory, "Victor Babes" National Institute of Pathology, Bucharest, Romania; Tel: +40213192732; Email:
Lupu AR, Cremer L, Popescu T (2015) Combined In vitro Effects of TiO2 Nanoparticles and Dimethyl Sulfoxide (DMSO) on HepG2 Hepatocytes. Int J Nanomater Nanotechnol Nanomed 1(1): 002-010. DOI: 10.17352/2455-3492.000002
© 2015 Lupu AR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
TiO2 nanoparticles; Hepatocytes; Toxicity; Solvents; Paints
BET: Brunauer-Emmett-Teller; DCFH-DA: 2'-7'-dichlorofluorescein-diacetate; DLS: Dynamic Light Scattering; DMEM-F12: Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12;DMSO: dimethyl sulfoxide; DRIFT: Diffuse Reflectance Infrared Fourier Transform Spectroscopy; EDX: Energy-dispersive X-ray Spectroscopy; FBS: Fetal Bovine Serum; FeHT: Fe3+-doped anatase TiO2 nanoparticles synthesized under hydrothermal conditions; HT: undoped anatase TiO2 nanoparticles synthesized under hydrothermal conditions; LD50: lethal dose 50%;MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; P25: Degussa P25 TiO2 nanoparticles; PBS: Phosphate-buffered Saline; ROS: Reactive Oxygen Species; TEM: Transmission Electron Microscopy; XRD: X-ray Diffraction
Introduction: Professional workers that manufacture or use titanium dioxide (TiO2)-based paints are exposed to potentially toxic TiO2 nanomaterials as well as to different paint solvents such as dimethyl sulfoxide (DMSO). In this context, we evaluate the combined cytotoxic effects of TiO2 nanoparticles and DMSO on HepG2 human hepatocytes.
Methods: Three types of TiO2 nanoparticles were used: commercial Degussa P25 and two samples synthesized by a hydrothermal procedure – undoped and Fe3+-doped TiO2. The effects of TiO2 nanoparticles on HepG2 cells exposed to DMSO before, after or together with the TiO2 treatment were investigated by viability and intracellular reactive oxygen species (ROS) determinations, performed using the MTT and DCFH-DA(2',7'-dichlorﬂuorescein-diacetate) methods respectively.
Results: Results indicated that DMSO made HepG2 cells more susceptible to toxic effects induced by nanosized TiO2. In the absence of DMSO, none of the tested nanoparticles exhibited significant cytotoxic effects. Viability increases were detected after 48 hours of treatment and attributed to possible redox-sensitive proliferation mechanism striggered by the low and moderate amounts of produced ROS. The combined action of TiO2 and DMSO led to a general viability decrease tendency. Significant effects (viability reductions and ROS generation) were observed in the case of cells first treated with Degussa P25 TiO2 and afterwards exposed to DMSO. The hydrothermal materials exhibited reduced in vitro reactivity on HepG2hepatocytes.
Conclusion: The study reveals the enhancement of nanosized TiO2 toxicity induced by DMSO exposure, its findings having potential to help in the evaluation of professional health risks associated to the combined action of TiO2 nanomaterials and paint solvents.
Recent studies regarding the widespread use of paints that contain titanium dioxide (TiO2, titania) nanoparticles for bacterial decontamination and self-cleaning purposes revealed significant advantages of these new technologies, but also potential health risks induced by the release of nanosized TiO2 from painted surfaces [1Kaiser JP, Roesslein M, Diener L, Wick P (2013) Human Health Risk of Ingested Nanoparticles That Are Added as Multifunctional Agents to Paints: an In vitro Study. PLoS ONE 8: e83215. doi:10.1371/journal.pone.0083215.-4Wang J, Fan Y (2014) Lung Injury Induced by TiO2 Nanoparticles Dependes on Their Structural Features: Size, Shape, Crystal Phases and Surface Coating. Int J Mol Sci 15: 22258-22278.]. If the amount of released TiO2 was shown to be relatively small under the studied conditions [5Al-Kattan A, Wichser A, Vonbank R, Brunner S, Ulrich A, et al. (2013) Release of TiO2 from paints containing pigment-TiO2 ornano-TiO2 by weathering. Environ Sci Process Impacts 15: 2186-93. doi:10.1039/c3em00331k.], significant professional health risks may be associated to workers that manufacture or use TiO2-based paints. These workers are exposed to potentially toxic TiO2 nanomaterials as well as different solvents used during the processing or application of such paints. Among the used solvents, dimethyl sulfoxide (DMSO) was considered to be more convenient due to its relatively reduced toxicity [6Marti M, Molina L, Aleman C, Armelin E (2013) Novel Epoxy Coating Based on DMSO as a Green Solvent, Reducing Drastically the Volatile Organic Compound Content and Using Conductory Polymers As a Nontoxic Anticorrosive Pigment. ACS Sustainable Chem Eng 1: 1609-1618.,77. Wu G (2010) Assay Development. Fundamentals and Practices. Chapter 13. High-Throughput Screening Assay Development. Wiley ISBN 978-0-470-19115-6, page 328.]. However, DMSO is also known to easily penetrate human skin and, in some cases, to serve as carrier agent, promoting the percutaneous absorption of other compounds (including drugs and toxins) [88. Capriotti K, Capriotti JA (2012) Dimethyl Sulfoxide. History, Chemistry and Clinical Utility in Dermatology. J Clin Aesthet Dermatol 5: 24-26.,9Botana LM (Ed) (2007) Phycotoxins: Chemistry and Biochemistry. Blackwell Publishing ISBN-13: 978-0-8138-2700-1, page 41.]. Under these circumstances, DMSO may facilitate the penetration of skin by TiO2 nanoparticles.
To evaluate the implications and hazards involved by the potential exposure to both, TiO2 nanoparticles and DMSO (or other paint solvent), it is essential to possess detailed knowledge regarding their combined effects on vital organs, such as brain, liver, heart, kidney, lung or spleen. Among these, the liver represents one of the most important organs involved in the processing of exogenous compounds (including nanomaterials) and detoxification, a significant amount of published studies concerning the hepatotoxic effects of solvents (including DMSO in several cases) [1010. Malaguamera G, Cataudella E, Giordano M, Nunnari G, Chisari G, et al. (2012) Toxic hepatitis in occupational exposure to solvents. World J Gastroenterol 18: 2756-2766.-1313. Mohammmadi S, Mehrparvar A, Labbafinejad Y, Attarchi MS (2010) The effect of exposure to a mixture of organic solvents on liver enzymes in an auto manufacturing plant. J Public Health DOI:10.1007/s10389-010-0340-z.].
The present study gives an insight into the combined in vitro effects of TiO2 nanoparticles and DMSO (seen as a generic paint solvent). The experiments have been performed on HepG2 cells (human hepatocarcinoma cells-ATCC® HB-8065™), a well characterized cell line, widely used in cytotoxicity studies due to its convenient specific characteristics, such as:
- biosynthetic capabilities similar to those of normal hepatocytes
- retainment of cell surface receptors – response capacity similar to normal cells [1414. Pinti M, Troiano L, Nasi M, Ferraresi R, Dobrucki J, et al. (2003) Hepatoma HepG2 cells as a model for in vitro studies in mitochondrial toxicity of antiviral drugs: which correlation with the pacient? J Biol Regul Homeost Agents 17: 166-71.,1515. Dehn P, White C, Conners D, Shipkey G, Cumbo T (2004) Characterization of the human hepatocellular carcinoma (HEPG2) cell line as an in vitro model for cadmium toxicity studies. In Vitro Cellular & Developmental Biology – Animal 40: 172-182.].
The studied effects concern the cytotoxicity and intracellular reactive oxygen species (ROS) production induced in HepG2 cells under different treatment schemes with nano-TiO2 and DMSO.
We have used three types of TiO2 nanoparticles: the commercial Degussa P25 TiO2 (P25) and other two samples synthesized under hydrothermal conditions in our laboratory – undoped (HT) and Fe3+-doped (FeHT) anatase TiO2. Degussa P25 TiO2 was often tested (and utilized as reference material) in studies concerning intracellular ROS generation [1616. Long TC, Tajuba J, Sama P, Saleh N, Swartz C, et al. (2007) Nanosize titaniumdioxide stimulates reactive oxygen species in brainmicroglia and damages neurons in vitro. Environ Health Perspect 115: 1631-1637.] and toxicity induced by titania nanomaterials [1717. Sayes CM, Wahi R, Kurian PA, Liu Y, West JL, et al. (2006) Correlating nanoscaletitania structure with toxicity: a cytotoxicity and inflammatoryresponse study with human dermal fibroblasts andhuman lung epithelial cells. Toxicol Sci 92: 174-185.-1919. Wadhwa S, Rea C, O’Hare P, Mathur A, Roy SS, et al. (2011) Comparative in vitro cytotoxicity study of carbon nanotubes and titania nanostructures on human lung epithelialcells. J Hazard Mater 191: 56-61.]. The hydrothermal TiO2 samples have similar structural characteristics (crystal structures, shapes, sizes and specific surface areas [2020. Popescu T, Lupu AR, Diamandescu L, Tarabasanu-Mihaila D, Teodorescu VS, et al. (2013) Effects of TiO2 nanoparticles on the NO2- levels in cell culture media analysed by Griesscolorimetric methods, J Nanopart Res 15: 1449-1450.] – the relevance of these factors being frequently considered in TiO2 nanotoxicology studies [2121. Jin C, Tang Y, Yang FG, Li XL, Xu S, et al. (2011) Cellular toxicity of TiO2 nanoparticles inanatase and rutile crystal phase. Biol Trace Elem Res 141: 3-15.,2222. Hsiao IL, Huang YJ (2011) Effects of various physicochemical characteristics on the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial cells. Sci Total Environ 409: 1219-1228.]), but different band gap energies (relative to each other) and colloidal behaviors (compared to Degussa P25) [20Popescu T, Lupu AR, Diamandescu L, Tarabasanu-Mihaila D, Teodorescu VS, et al. (2013) Effects of TiO2 nanoparticles on the NO2- levels in cell culture media analysed by Griesscolorimetric methods, J Nanopart Res 15: 1449-1450.].
The obtained results are analyzed with respect to the structural and physicochemical properties of the tested nanomaterials [20Popescu T, Lupu AR, Diamandescu L, Tarabasanu-Mihaila D, Teodorescu VS, et al. (2013) Effects of TiO2 nanoparticles on the NO2- levels in cell culture media analysed by Griesscolorimetric methods, J Nanopart Res 15: 1449-1450.], the characteristics of the used cells and the cell penetration and hydroxyl radical scavenger properties of DMSO [2323. Mokudai T, Nakamura K, Kanno T, Niwano Y (2012) Presence of Hydrogen Peroxide, a Source of Hydroxyl Radicals, in Acid Electrolyzed Water. PLoS ONE 7: e46392. doi:10.1371/journal.pone.0046392.].
Materials and Methods
The undoped and iron-doped TiO2 nanoparticles were prepared starting from TiCl3 (solution 15 % in HCl 10 %, from Merck) and Fe2O3 (RITVERC 95.44 % 57Fe Isotopic Enrichment) [20Popescu T, Lupu AR, Diamandescu L, Tarabasanu-Mihaila D, Teodorescu VS, et al. (2013) Effects of TiO2 nanoparticles on the NO2- levels in cell culture media analysed by Griesscolorimetric methods, J Nanopart Res 15: 1449-1450.].
To obtain the Fe3+ (1 at. %)-doped TiO2, the titanium and iron precursors were processed as follows: TiCl3 was oxidized (by air barbotage) to TiCl4 and Fe2O3 was reacted to hydrochloric acid (4N) to form FeCl3. The resulted solutions were involved in a coprecipitation process, NH4OH being drop wise added to their mixture up to pH=8. The obtained precipitate was washed with deionised water, resuspended in double-distilled water and exposed to hydrothermal treatment in a 50 cm3 Teflon-lined autoclave at 200 °C for one hour. The undoped TiO2 was synthesized using the same procedure involving the titanium precursor only.
The structural, morphological, optical and physicochemical characteristics of the used materials have been studied by X-ray diffraction (XRD), transmission electron microscopy (TEM), Energy-dispersive X-ray spectroscopy (EDX), Mossbauer spectroscopy, Brunauer-Emmett-Teller (BET) nitrogen adsorption, UV-Vis reflectance spectroscopy, Dynamic Light Scattering (DLS) and Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFT) and described in a previous work [20Popescu T, Lupu AR, Diamandescu L, Tarabasanu-Mihaila D, Teodorescu VS, et al. (2013) Effects of TiO2 nanoparticles on the NO2- levels in cell culture media analysed by Griesscolorimetric methods, J Nanopart Res 15: 1449-1450.].
Cellular and noncellular experiments regarding the in vitro effects of TiO2 nanoparticles on HepG2 hepatocytes pre-treated, co-treated or post-treated with DMSO
Cell viabilities and intracellular ROS productions in HepG2 hepatocyte cultures exposed to TiO2 nanoparticles and DMSO have been performed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and the DCFH-DA (2',7'-dichlorﬂuorescein-diacetate) test respectively.
The experiments were such designed to elucidate the following aspects:
- effects of the studied TiO2 nanoparticles on hepatocytes that were “already damaged” – the cells were first treated with DMSO and two hours later exposed to the action of nano-TiO2;
- viability and intracellular ROS production in case of HepG2 cells simultaneously treated with DMSO and nano-TiO2;
- response of hepatocytes to the action of DMSO, administered two hours after the cells were exposed to nano-TiO2;
- The working protocol was established based on the following: LD50 for DMSO after 24 h of exposure – the DMSO dose capable of killing 50% of the cells after 24 h of exposure;
- the effects of nano-TiO2 alone (without DMSO) on the studied cells;
The HepG2 cells were seeded in 24-well culture plates at a density of 105 cells/cm2 in volumes of 1 ml of DMEM-F12 culture medium containing 10% FBS. After 24 h required for cell adherence and growth, the culture medium was discarded and replaced with fresh medium (1 ml/well) containing the necessary treatment agents (TiO2 (2.5, 7.5, 15, 25, 50, 75, 100 μg/ml), DMSO (LD50) or both) according to the stimulation scheme presented above. In case of pre-treatment or post-treatment with DMSO, either TiO2 or DMSO, were added two hours after the initial stimulation.
Preliminary experiments have established the LD50 for DMSO to be 5 μl DMSO/1 ml of culture medium.
Cell viability assay
After 24, 48 and 72 h of treatment, the culture medium was removed and MTT solution (1 mg/ml MTT in phosphate buffered saline (PBS)) was added to each well (300µl/well). The obtained samples were incubated for 2 hours, the MTT solution being afterwards discarded. To dissolve the produced formazan crystals, DMSO (Sigma-Aldrich) was added to each well (300µl/well). The optical density of the purple formazan solution was determined at 540 nm using a Thermo Multiskan EX spectrophotometer.
The obtained results were quantified with respect to control samples consisting of untreated cells (in case of experiments in which TiO2 alone was used) or cells treated with DMSO (in case of experiments involving both TiO2 and DMSO). Cell viability was expressed as percents versus control. Standard deviations were computed based on three technical replicates corresponding to each sample and three independent biological replicates of each experiment.
The results are represented as average values +/- standard deviations (error bars).
Determination of intracellular ROS production
To quantify the intracellular ROS production, the cells were incubated for 24 hours with the treating agents (TiO2 (2.5, 25, 50, 100 μg/ml), DMSO (LD50) or both). After incubation, the culture medium was removed and replaced with fresh medium containing DCFH-DA (0.2 μl of 2'-7'-dichlorofluorescein-diacetate (DCFH-DA) stock solution (25 mg/mL DCFH-DA in TFS) in 1 ml of culture medium). The obtained samples were incubated again for 30 minutes. The DCFH-DA medium was afterwards removed; the cells were detached with trypsin (0.25% trypsin and 0.53 mM EDTA solution), suspended in PBS and centrifuged for 10 min at 1500 rpm and 4°C, the supernatant being discarded. The excess of fluorescein was removed by washing the cells twice in PBS and the cells were resuspended in 500 μl PBS. The homogenized suspensions were transferred to 96-well plates (100μl/well). A Fluoroskan FL (Thermo) equipment (excitation wavelength 485 nm/emission wavelength 530 nm) was used to perform the fluorimetric determinations.
The obtained results were quantified with respect to control samples (described above for cell viability experiments) and expressed as percents versus control. Standard deviations were computed based on three technical replicates corresponding to each sample and three independent biological replicates of each experiment.
The results are represented as average values +/- standard deviations (error bars).
Data analysis and representation
Data statistical analysis and representation were performed using the Sigma Plot-11 software package. Depending on data normality, either one-way ANOVA or one-way ANOVA on ranks tests were performed. The Student–Newman–Keuls (SNK) posthoc test was employed in order to complete the analysis. A value of p < 0.05 was considered significant. All samples statistically different from controls were marked on figures with a (*).
The detailed structural and physicochemical characterization of the three TiO2 nanomaterials used in this study was published in a previous work [20Popescu T, Lupu AR, Diamandescu L, Tarabasanu-Mihaila D, Teodorescu VS, et al. (2013) Effects of TiO2 nanoparticles on the NO2- levels in cell culture media analysed by Griesscolorimetric methods, J Nanopart Res 15: 1449-1450.]. Briefly, all three types of titania have similar shapes (no acicular shaped particles) and average particle sizes between 10-30 nm. The hydrothermal, HT and FeHT, samples have anatase structure and Degussa P25 TiO2 is a mixture of anatase and rutile polymorphs with anatase/rutile weight ratio of 85:15(%). BET specific surface areas are 49 m2/g for Degussa P25, 130.62 m2/g for HT and 114.81 m2/g for FeHT. The band gap energies were approximately 3 eV for Degussa P25 and HT and 2.848 eV for the iron doped, FeHT, sample. Degussa P25 has considerably higher colloidal stability in aqueous suspensions compared to HT and FeHT. The colloidal stabilization effect of proteins from culture medium was revealed.
In vitro effects of undoped and Fe3+-doped TiO2 nanoparticles on HepG2 hepatocytes
TiO2 alone: Cell viabilities obtained in experiments involving TiO2 alone (no DMSO) are represented in Figure 1a-1c. No significant viability variations were observed after 24h and 72h of treatment for none of the tested TiO2 samples. At 48h after exposure, viability increases (up to 49 %) were detected for all nanomaterials, being more pronounced in case of FeHT. The observed increases were proportional to the concentration of TiO2 in the studied samples.
To better illustrate the specific features of each experimental case (pre-, co- or post-treatment with DMSO) and each type of tested TiO2, the results are described and discussed comparatively bellow.
TiO2 and DMSO: While for all the tested materials, the most prominent viability reductions were observed in the case of cells post-treated with DMSO (TiO2-DMSO) (Figures 2a-4a), the highest cell killing effect was induced by Degussa P25 nanoparticles (Figure 2a). The hydrothermal materials (HT and FeHT) induced only weak or insignificant cytotoxic effects.
The observed viability variations did not depend on the treatment time (24, 48, 72 hours), for none of the tested TiO2 types.
Intracellular ROS production
TiO2 alone: Regarding the intracellular ROS production, significant increases (between 21- 39 %) were induced by the iron-doped sample (Figure 1d). The commercial P25 TiO2 induced a significant increase only at its maximum concentration (100 µg/ml). The observed increases were concentration-dependent. No variation was detected in case of HT sample (Figure 1d).