Inhalation Toxicology, 2009; 21(S1): 115–122
RESEARCH ARTICLE
Carbon black and titanium dioxide nanoparticles induce pro-inflammatory responses in bronchial epithelial cells: Need for multiparametric evaluation due to adsorption artifacts Stéphanie Val1, Salik Hussain1,2, Sonja Boland1, Rodolphe Hamel1, Armelle Baeza-Squiban1, and Francelyne Marano1 Univ ParisDiderot-Paris 7, Unit of Functional and Adaptive Biology (BFA) EAC CNRS 7059, Laboratory of Molecular and Cellular Responses to Xenobiotics, France, and 2Department of Pathology, University of Veterinary and Animal Sciences Lahore, Pakistan
1
Abstract The initiation of an inflammatory process is the main adverse effect observed following the exposure of the airway epithelium to nanoparticles (NPs). This study was designed to explore the pro-inflammatory potential of two different NPs of similar size but of different compositions (CB 13 nm and TiO2 15 nm) on a human bronchial epithelial cell line (16HBE14o-). The expression of granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin (IL-6), and tumor necrosis factor alpha (TNFα) was evaluated in terms of mRNA, intracellular proteins, and released cytokines. Exposure to NPs induced a dose-dependent expression of all these cytokines, depending upon the chemical composition of NPs. The released cytokines appeared to be an inaccurate methodology to evaluate the pro-inflammatory response. Indeed, NPs adsorbed cytokines, and the binding was dependent on the nature of both the cytokine and NPs. Furthermore, addition of fetal calf serum or bovine serum albumin improved the detection of cytokines but also reduced cellular responses. Use of different detergents (Tween, Triton, and NP40) demonstrated limited efficiency to desorb cytokines from NPs. Thus, this study demonstrated the pro-inflammatory potential for CB and TiO2 NP but underlines the methodological artifacts faced during the in vitro evaluation of cytokine release that necessitates a multiparametric evaluation. Keywords: Nanoparticles; cytokines; 16HBE; protein adsorption; inflammation
Introduction Nanotechnologies have undergone a revolution in the recent past, as many sophisticated methodologies have been developed to study and manipulate matter at the nanoscale. Increasing utilization of nanomaterials in industrial as well as consumer products has increased the possibilities of human exposures. Due to this fact, risk assessment of nanomaterials has become crucial. Nanoparticles (NPs) are present in the different compartments of the environment and hence can enter the organisms by contact, ingestion, and inhalation. However, inhalation is the most important route of entry in the case
of unintentional exposure. Experimental studies in animals have shown that NPs induce pulmonary inflammation (Oberdörster et al., 2000; Renwick et al., 2004; Oberdörster et al., 2005). Persistence of insoluble NPs in the respiratory tract can trigger chronic inflammation—the hallmark of different respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD)—and NP-induced acute pulmonary inflammation could act as an exacerbating factor for these respiratory diseases. The ability of NPs to induce pulmonary inflammation has been attributed to their increased surface reactivity as compared to micrometric counterparts (Donaldson et al., 2005). This high surface
S. Hussain and S. Val contributed equally to this work. Address for Correspondence: S. Hussain, Univ ParisDiderot-Paris 7, Unit of Functional and Adaptive Biology (BFA) EAC CNRS 7059, Laboratory of Molecular and Cellular Responses to Xenobiotics, Case 7073, 5 rue Thomas Mann, 75 205 Paris cédex 13, France. E-mail:
[email protected] (Received 26 March 2009; accepted 02 April 2009) ISSN 0895-8378 print/ISSN 1091-7691 online © 2009 Informa UK Ltd DOI: 10.1080/08958370902942533
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116 S. Val et al. reactivity of NPs plays a key role in their biological effects that vary from pro-inflammatory response to cell death due to their potential to produce reactive oxygen species (ROS). The large surface area characterizing NPs can also be involved in biochemical reactions with proteins, which could result in functional changes in the constituents of the fluids lining the airways (Kreyling et al., 2007). Moreover, protein coating of NPs could modify their uptake and/or transcytosis. The adsorption of biologically important molecules on NP surfaces could not only lead to altered cellular responses but could also interfere with the measurements of toxicological endpoints classically used to characterize biological effects. Keeping in view all these considerations, the present study was designed to investigate the ability of carbon black (CB) and titanium dioxide (TiO2) NPs to induce a pro-inflammatory response in a human bronchial epithelial cell line (16HBE14o-). This pro-inflammatory response was evaluated in terms of release as well as intracellular production and mRNA expression of three different proinflammatory cytokines: interleukin (IL-6), granulocyte monocyte colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF). Furthermore, the ability of NPs to adsorb these cytokines, the ability of bovine serum albumin (BSA) and fetal calf serum (FCS) to prevent cytokine binding, and finally the desorption of cytokines from NPs by the use of detergents were investigated. In addition, the impact of using BSA and FCS was evaluated in terms of cytotoxicity and pro-inflammatory potential of NPs. Our results indicate that CB and TiO2 NPs are able to induce a pro-inflammatory response at non-cytotoxic concentrations that cannot be accurately demonstrated by the sole measurement of cytokine release since NPs bind cytokines. However, this binding differs with the chemical nature of NPs and depends on the cytokine under investigation. This cytokine adsorption onto NPs can be modulated by using BSA and FCS, but this leads to decreased pro-inflammatory as well as cytotoxic potentials of NPs.
Materials and methods All the chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) unless otherwise specified. Particles Stock suspensions of CB 13 nm (BET surface area 350 m² g−1; Degussa, Frankfurt, Germany) and TiO2 15 nm (BET surface area 190–290 m² g−1; anatase 99.9%) were made at a concentration of 2 mg ml−1 in DMEM/F12 media (Dulbecco Modified Eagle Medium Nutrient Mix F-12, Invitrogen, Cergy-Pontoise, France) after a sonication of 3 min at 60W (induced ultrasonic probe, Fisher Bioblock Scientific, Illkirch, France) and stored at −20°C until use. Before utilization, NPs were sonicated 3 times for 20 s at 60W and diluted in cell culture medium without Ultroser G.
Cells and culture conditions The human bronchial epithelial cell line 16HBE14o(a kind gift from Dr. Gruenert, National Institutes of Health [NIH], San Francisco, CA) was grown in DMEM/F12 cell culture media supplemented with penicillin (100 µg ml−1), streptomycin (100 µg ml−1), fungizone (1 µg ml−1), glutamine (0.292 µg ml−1), and Ultroser G (UG) 2% (Biosepra; Cergy Pontoise, France). Cells were cultured on plastic material (Costar, VWR; Fontenay-Sous-Bois, France) after coating with type I collagen at 4 µg cm−2 and were maintained in an incubator at 37°C, under 5% CO2. Except otherwise specified, cells were seeded in 12-well plates at a density of 10,000 cells cm−2, and after 72 h of growth, they were grown for a further 24 h in UG-free media before treatment with NPs. WST-1 assay Water-soluble tetrazolium salt assay (2-(4-iodophenyl)-3(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium; Roche Diagnostics; Mannheim, Germany) was used to evaluate the metabolic activity of the cultures. Cells were cultured in 96-well plates at a density of 20,000 cells/well and were allowed to grow for the next 48 h. After 24 h of culture in UG-free media, cells were treated with NPs in DMEM/F12 media without phenol red for a further 24 h. At the end of the exposure, culture medium was removed, and cells were rinsed and incubated with the WST-1 reagent for 2 h at 37 °C. Finally, optical density was measured at 450 nm wavelength with the help of a plate reader (DYNEX MRX 5000, Dynatech Laboratories, Chantilly, VA). It was confirmed beforehand that there is no interference between the optic density of WST-1 reagent and the presence of NPs. Cytokine assay After subconfluent cultures had been exposed to the NPs for 24 h, supernatants were recovered, centrifuged at 10,000 × g for 10 min at 4°C, and cells were incubated with 1% Nonidet P (NP 40) and 1% protease inhibitor mixture for 1 h at 4°C under a gentle rotation. The protein extract was recovered, centrifuged at 15,000 × g for 15 min at 4°C. Supernatants and extracts were frozen at −80°C until further analysis. The concentrations of GM-CSF, IL-6, and TNF- released into the culture supernatant were evaluated with a commercially available human enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Europe; Abingdon, UK) according to the manufacturer’s recommendations. Cytokine adsorption assay Cytokine binding capacity of CB and TiO2 NPs was evaluated at similar concentrations as used for the evaluation of their pro-inflammatory potential, i.e., 25 µg ml−1 and 50 µg ml−1, respectively. Cytokine standards provided in the ELISA kits were utilized to prepare working solutions of 250 pg ml−1 in DMEM/F12 medium. This cytokine solution was incubated with NPs for 24 h (37°C, 5% CO2) supplemented or not with 0.5–5% FCS or 0.03–0.3% BSA in 48-well culture plates. After incubation, suspensions were collected, centrifuged at 10,000 × g for
10 min at 4°C, and concentration of cytokines in the supernatant was evaluated by ELISA kit. In order to limit adsorption of cytokine onto the plastic plates, the cell culture plates were pretreated with either collagen type 1 (4 µg cm−2) or BSA (0.3%) for 5 min and rinsed 3 times with cell culture medium before co-incubation of NPs and cytokines. Cytokine desorption NPs were mixed with cytokines and incubated for 24 h as described for the cytokine adsorption assay. After incubation, different detergents (Tween 20, Triton X100, and NP40) were added at 0.01–0.5% and further incubated for 30 min with regular vortexing each 5 min. NPs were centrifuged at 10,000 × g for 10 min at 4°C and cytokines were detected in the supernatant by ELISA as described previously. Real-time quantitative polymerase chain reaction (qPCR) Polymerase chain reaction (PCR) was performed to evaluate GM-CSF, IL-6, and TNF mRNA expression. Ribosomal protein L19 (RPL19) gene was used as an internal control. Cells were treated with NPs of CB or of TiO2 (1–10 µg cm−2) for 24 h, and mRNA extraction and purification was performed by a commercially available kit (SV Total RNA Isolation System, Promega; Charbonnières-les-bains, France) according to the manufacturer’s recommendations. Reverse transcription was done by M-MLV Reverse Transcriptase kit (Promega, Charbonnières-les-bains, France). Real-time qualitative PCR (qPCR) analysis was performed using MX 3000P (Stratagene, Amsterdam, Netherlands). The following primer sequences were used: RPL 19: Sense: 5-GGC TCG CCT CTA GTG TCC TC-3. Anti-sense: 5-CAA GGT GTT TTT CCG GCA TC-3. GM-CSF: Sense: 5-AGC CGA CCT GCC TAC AGA C- 3. Anti-sense: 5-GGG CAG TGC TGC TTG TAG TG-3. IL-6: Sense: 5-ACA GCC ACT CAC CTC TTC AG-3. Anti-sense: 5-TGG AAG CAT CCA TCT TTT TC-3. TNF: Sense: 5-GCT CCC CAA GAA GAC AGG- 3. Anti-sense: 5-GCC AGA GGG CTG ATT AGA G-3. The relative quantification of the gene of interest was done according to the method described by Pfaffl (2001). Statistical analysis Data represented as mean ± SD were evaluated by analysis of variance (ANOVA) followed by Dunnet’s t-test to examine the differences between the different treated groups with respect to control, whereas the Newmann–Keuls test was used to evaluate the difference among the treated groups, with p
