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Assessing the respiratory toxicity of dihydroxyacetone using an in vitro human airway epithelial tissue model
Toxicology in Vitro 59 (2019) 78–86
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Assessing the respiratory toxicity of dihydroxyacetone using an in vitro human airway epithelial tissue model Yiying Wanga, Qiangen Wub, Levan Muskhelishvilic, Kelly Davisc, Matthew Bryantb, Xuefei Caoa,
T ⁎
a
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States of America Division of Biochemistry Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States of America c Toxicologic Pathology Associates, Jefferson, AR 72079, United States of America b
A R T I C LE I N FO
A B S T R A C T
Keywords: In vitro human air-liquid-interface (ALI) airway epithelial tissue model Dihydroxyacetone (DHA) Respiratory toxicity Cloud liquid aerosol generation and exposure system
Dihydroxyacetone (DHA) is an approved color additive used in sunless tanning lotions. Recently, there has been an increased use of DHA in sunless tanning booths in a manner that could result in its inhalation during application. In the present study, we have evaluated the potential for DHA causing toxicity via inhalation using a human air-liquid-interface (ALI) in vitro airway epithelial tissue model. ALI airway models have a close structural and functional resemblance to the in vivo airway epithelium, and thus data generated in these models may have relevance for predicting human responses. To simulate in vivo exposure conditions, we employed a method for liquid aerosol generation that mimics the physical form of inhaled chemicals and used doses of DHA and an exposure frequency reflecting human respiratory exposures during tanning sessions. Compared to the vehicle control, cilia beating frequency (CBF) and MUC5AC secretion were significantly decreased after each exposure. However, time-course studies indicated that both CBF and MUC5AC secretion returned to normal levels within 3 days after the treatment. Matrix metalloproteinase (MMP) release, on the other hand, was decreased 24 h after the first exposure and its level returned to baseline after 5 exposures. No significant morphological changes occurred in the DHA-treated cultures after 5 weekly exposures. Our findings indicate that DHA, at concentrations likely to be experienced by humans, has transient toxic effects on human airway ALI cultures.
1. Introduction Inhalation exposures to toxicants are associated with asthma, chronic obstructive pulmonary disease (COPD), and lung cancer (Barnes, 2008; BéruBé et al., 2009; Ghorani-Azam et al., 2016; Knight and Holgate, 2003; Pope et al., 2002). The lung serves both as a target tissue and portal of entry for toxicants into the systemic circulation. Characterizing adverse responses in airway epithelium, therefore, could provide valuable information for evaluating health hazards from inhalation exposures. Traditionally, rodent models are considered the gold standard for inhalation toxicology studies. However, critical differences in dosimetry, geometry, and physiology between rodent and human respiratory systems raise concerns over extrapolating human responses from rodent test data. Furthermore, the cost and time involved in conducting in vivo inhalation testing make it challenging to obtain inhalation toxicity data on the large number of new and existing chemicals, respiratory toxicants, and inhaled therapeutics (Costa, 2008). Together with the increasing pressure from animal rights groups, it is desirable to develop alternative human-based in vitro
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approaches for inhalation toxicity screening and testing. A number of in vitro lung models are available and have been used for inhalation studies (BéruBé et al., 2009). Among them, the human in vitro air-liquid-interface (ALI) airway epithelial tissue model has received increasing attention for its potential applications in inhalation toxicology (BéruBé et al., 2009; Cao et al., 2015, 2017; Chortarea et al., 2015). The well-differentiated ALI airway tissue model is derived from primary human tracheobronchial epithelial cells cultured at the air-liquid interface. Unlike conventional monolayer cell lines generated from a single cell type, the ALI tissue model consists of three major airway epithelial cell types, i.e., ciliated cells, goblet cells, and basal cells, arranged in a pseudostratified structure. Key in vivo functions, such as tight junctions and trans-epithelial electrical resistance, are retained (Cao et al., 2015, 2017). Genetic and proteomic analyses have further revealed a strong resemblance between the transcriptome and secretome of ALI models and human airway epithelium (Dvorak et al., 2011; Kesimer et al., 2009). Besides the structural and functional similarities, the presence of the unique air-liquid interface of the ALI cultures makes direct exposures to aerosols, vapors, and particles possible. Thus, the
Corresponding author at: U.S. FDA/NCTR, Division of Genetic and Molecular Toxicology, 3900 NCTR Road, Jefferson, AR 72079, USA. E-mail address: [email protected] (X. Cao).
https://doi.org/10.1016/j.tiv.2019.04.007 Received 15 February 2019; Received in revised form 3 April 2019; Accepted 4 April 2019 Available online 05 April 2019 0887-2333/ Published by Elsevier Ltd.
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cells at 37 °C during the exposures (Fig. 1). The operational principal of the Cloud System has been described by Lenz et al. (Lenz et al., 2014). 1,3-Dihydroxyacetone (DHA, Toronto Research Chemicals, North York, Canada) was dissolved in diluted Dulbecco's Phosphate-Buffered Saline (DPBS, pH 7.4, Corning, Manassas, VA; 1:80 diluted in molecular grade water) to concentrations of 0.2, 0.4, and 1.0 M. To quantify aerosol deposition, 24-well PET Transwell® cell culture inserts (Corning, Tewksbury, MA) containing 100 μL DPBS were placed in all eight positions of the exposure module pre-warmed to 37 °C. 1 mL DHA solution was added to the nebulizer and immediately nebulized for 30 s. The exposure chamber was removed 20 min after the nebulization to ensure complete settlement of the aerosols onto the culture inserts. 90 μL DPBS containing the deposited DHA were collected from each insert for chemistry analysis. DHA was quantified using a method described by Biondi et al. (2007) with a few modifications. Briefly, 10 μL of standards or samples were derivatized for 30 min at 65 °C in 5 mg/mL O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride (PFBHA, Sigma-Aldrich, St. Louis, MO) prepared in 10 mM ammonium acetate solution (pH 4.0). The reaction mixture was cooled down on ice and diluted in 4 volumes of acetonitrile. 10 μL dilutions were injected into an HPLC equipped with a Waters 2695 separation module connected to an Atlantis T3 column (4.6 mm × 150 mm, 5 μm) and a Waters ACQUITY QDa Mass Detector. The derivatization product was eluted in an isocratic elution using a mobile phase composed of water (Mobile A) and HPLC-grade acetonitrile (Mobile B), both spiked with 0.1% formic acid. Elution was conducted with 80% of Mobile B at a flow rate of 0.5 mL/min for 5 min. The temperature of the HPLC column was set at 40 °C. Elution of DHA was monitored at m/z 286.0. The amount of DHA was calculated using a linear standard curve with a concentration range from 1.6 to 1000 μg/ mL (R2 = 0.99). The deposition factors for DHA aerosols were calculated using the following formula: deposition factor = (DHA deposition)measured/(DHA deposition)target × 100%, where measured DHA deposition was the mass of DHA quantified in each insert using chemical analysis and target DHA deposition was defined as ([DHA]working solution × nebulization volume) × (surface area of each insert/surface area of the exposure module).
toxicity of inhaled toxicants can be evaluated in these tissue models using bioassays reflecting tissue responses and under conditions mimicking in vivo inhalation exposures. DHA is an approved color additive used in sunless tanning lotions (U.S. Food and Drug Administration, 2016). Its glycosidic hydroxyl group reacts with amino groups of epidermal proteins via non-enzymatic glycation, giving the skin a brownish color as the result of the formation of melanoidins (Gallagher, 2018; Mullarkey et al., 1990; Namiki, 1988). DHA use is restricted to external applications; because relevant safety data are lacking, its contact with body surface covered by mucous membranes is strongly discouraged (Office of Cosmetics and Colors et al., 2003; Scientific Committee on Consumer Safety, 2014; U.S. Food and Drug Administration, 2016). However, unintentional exposure of respiratory mucosal surfaces to DHA aerosols is highly possible during sunless tanning sessions, a possibility for which it is not approved (Scientific Committee on Consumer Safety, 2014). In this study, we evaluated the potential respiratory toxicity of DHA using the ALI airway tissue model. To mimic in vivo exposure conditions, we used a liquid aerosol generation and exposure system (Chortarea et al., 2015; Lenz et al., 2014) and exposed ALI cultures to a range of non-cytotoxic doses of DHA aerosols at the air-liquid interface. A weekly treatment regimen was employed to mimic the tanning frequency of consumers. ALI cultures were repeatedly exposed to DHA aerosols once per week for up to 5 weeks. A panel of endpoints related to tissue responses was assessed at several time points after each exposure to understand the temporal response and recovery of the ALI cultures. 2. Materials and methods 2.1. Quantification of DHA aerosol deposition The design of the Vitrocell® Cloud 12/12 Liquid Aerosol Exposure System (Waldkirch, Germany) is schematically illustrated in Fig. 1. The Cloud System consists of a nebulizer with a perforated, piezoelectrically driven vibrating membrane (Aeroneb® Pro, Aerogen, Galway, Ireland), a removable exposure chamber for confining the aerosols, an exposure module with an integrated quartz crystal microbalance (QCM) for realtime monitoring of mass deposition, and a heating unit for maintaining
2.2. Human ALI airway tissue model Human ALI airway tissue models were established using the PneumaCult™-ALI Medium kit (STEMCELL Technologies, Vancouver, Canada) as described previously (Cao et al., 2017). Briefly, normal human primary tracheobronchial epithelial cells (The CF Center Tissue Procurement and Cell Culture Core, University of North Carolina, Chapel Hill, NC) were expanded on 100-mm tissue culture dishes in PneumaCult™-Ex Expansion Medium (STEMCELL Technologies) until cells reached approximately 80% confluence. Cells were collected by trypsinization and resuspended in the Expansion Medium at a density of 4 × 105 cells/mL; 100 μL cell suspensions were added to each 24-well PET Transwell® cell culture insert. Expansion medium was added to both the apical and basolateral compartments to support cell proliferation. When cells grew to 100% confluence, the medium in the apical compartment was removed and cells were fed from the basolateral side only with PneumaCult™-ALI Maintenance Medium every other day for 4 weeks until they develop the structure of pseudostratified ciliated epithelium. Fig. 1. Schematic diagram of the VITROCELL® Cloud 12 Liquid Aerosol Exposure System. The Cloud 12 consists of an exposure chamber, an exposure module, and an electronically controlled heating unit. A Quartz Crystal Microbalance (QCM) Sensor is installed at the A1 position of the exposure module for real-time dose monitoring. The exposure chamber is divided into two unequal compartments, each equipped with a vibrating mesh nebulizer on the top. Such a setup allows concurrent exposures to the vehicles and test articles.
2.3. DHA aerosol exposure Dulbecco's Modified Eagle's Medium (DMEM, American Type Culture Collection, Manassas, VA), supplemented with 27 mM 4-(2hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) and 0.1 mM sodium pyruvate (Life Technologies, Grand Island, NY), was used as the exposure medium. 3.5 mL of pre-warmed media were added to each 79
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lysates were coated on a high-protein-binding ELISA plate (Thermo Fisher Scientific). The ELISA plates were first blocked with bovine serum albumin (BSA; 10 mg/mL) for 1 h at room temperature, followed by a 1-h incubation with anti-MUC5AC (45 M1) (Pierce) or anti-MUC5B antibodies (Abcam, Cambridge, MA) (1:500 dilution in DPBS containing 10 mg/mL BSA, 0.3% Triton X-100, and 0.2% Tween-20). 100 μL HRPconjugated goat-anti-mouse antibody (1:1000 dilution in DPBS containing 10 mg/mL BSA, 0.3% Triton X-100, and 0.2% Tween-20; Rockland, Limerick, PA) then were added to each well and incubated for 30 min. Excess secondary antibodies were removed by washing with DPBS three times. Color was developed by adding 50 μL of TMB substrate (Thermo Fisher Scientific) to each well; color reactions were stopped with 50 μL of 2 N HCl. The absorbance at 450 nm was measured using a Synergy H4 microplate reader. Raw data are presented in Supplementary Tables 2 to 4.
well in the pre-warmed exposure module; culture inserts were hung in each well by the plastic rim. The basolateral side of the inserts touched the exposure medium and the apical side was exposed to air for aerosol exposures. DHA aerosols were generated as described in Quantification of DHA aerosol deposition. Upon conclusion of the exposure, only the basolateral sides of the cultures were washed with DPBS, leaving the apical surface undisturbed; 400 μL fresh Maintenance Media were added to the basolateral compartment. The cultures were maintained in a humidified incubator at 37 °C with 5% CO2. By handling cultures this way, cells were continuously exposed to DHA aerosols from the apical side until the first mucus collection that occurred at 24 h after the exposure. Spray tan from one tanning session usually lasts for 5 to 7 days (Garone et al., 2015; Levy, 1992). Spray tanning, therefore, typically is conducted every 7 days to maintain the color. To mimic the tanning frequency by consumers, ALI cultures were exposed to DHA aerosols once per week for up to 5 weeks.
2.8. Matrix metalloproteinase (MMP) Bio-plex assay
2.4. Adenylate kinase (AK) cytotoxicity assay
Secretion of select MMP proteins into the basolateral media was quantified using the Bio-plex Pro Human MMP 9-plex assay kit and a MAGPIX system (Bio-Rad, Hercules, CA). 50 μL basolateral media collected 24 h after the first and fifth exposures were assayed. The assays were performed by following the manufacturer's instructions. Concentrations of the MMP proteins were calculated using a concurrent standard and Bio-Rad Bio-Plex Manager software (version 4.1). Raw data are presented in Supplementary Table 5.
The cytotoxicity of DHA aerosols was assessed by measuring the release of intracellular Adenylate Kinase (AK) from damaged cells using a ToxiLight™ Non-destructive Cytotoxicity BioAssay Kit (Lonza, Walkersville, MD). 24 h after the exposure, the apical side of the cultures was washed twice with 100 μL DPBS and the apical washes were combined into a microcentrifuge tube; basolateral medium was collected into a separate microcentrifuge tube. For the AK cytotoxicity assay, 70 μL basolateral media and 35 μL apical washes were combined and incubated with the AK Detection Reagent for 5 min. Luminescence was measured using a Synergy H4 microplate reader (BioTek, Winooski, VT). Total AK release was determined by assaying basolateral medium from cultures treated with 1% Triton™ X-100 for 24 h at 37 °C. Results are presented as percent AK release.
2.9. Histology The ALI cultures were washed briefly with DPBS and fixed in 10% neutral buffered formalin for 48 h. The membranes were routinely processed and embedded in paraffin. 5-μm thick tissue sections were cut, mounted on positively charged slides, and deparaffinized and hydrated by processing through xylene and graded ethanol solutions. Hematoxylin and eosin (H&E) staining was conducted using a Leica Autostainer. For evaluation of the apoptotic index, deparaffinized tissue sections were immunohistochemically stained with a cleaved caspase-3 antibody. Briefly, the cleaved caspase-3 antigen was retrieved by boiling the tissue sections in 0.01 M citrate buffer, pH 6.0, for 15 min (3 × 5 min) using a microwave oven. The slides were washed in H2O for 5 min after cooling to room temperature. Endogenous peroxidase was then quenched by treating tissue sections with 3% hydrogen peroxide containing 0.1% sodium azide for 10 min. After blocking the nonspecific binding in 0.5% casein for 20 min, the slides were incubated for 1 h with the rabbit polyclonal cleaved caspase-3 antibody (1:200 dilution in DPBS containing 1% BSA; Cell Signaling, Danvers, MA), followed by sequential incubation with biotinylated goat-antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and ExtrAvidin-conjugated horseradish peroxidase (Sigma-Aldrich) at dilutions of 1:200 and 1:100, respectively. Staining was developed with diaminobenzidine substrate (DAB; Sigma-Aldrich) for 5 min. Sections were then counter-stained with hematoxylin and mounted with Permount™ Mounting Medium (Thermo Fisher Scientific). Rabbit IgG was used in place of primary antibody to serve as the negative control. For evaluating goblet cell hyperplasia/hypoplasia, a separate set of sections was histochemically stained with the periodic acid Schiff (PAS) reaction. PAS staining labels glycoprotein and glycolipid components in mucins. It is widely used for identifying mucus-secreting goblet cells and assessing the morphology of goblet cells. The slides were incubated in periodic acid solution for 5 min at room temperature. The slides were then washed with tap water and incubated in Schiff's reagent for 30 min at room temperature, followed by additional washing in running tap water. Sections were counter-stained with hematoxylin and coverslipped with Permount™ Mounting Medium.
2.5. Trans-epithelial electrical resistance (TEER) TEER was measured using an EVOM2 epithelial volt-ohmmeter and an STX2 electrode (World Precision Instruments, Sarasota, FL). Changes in TEER values indicate the effect of treatments on paracellular barrier properties. The EVOM2 was calibrated using a test resistor prior to the measurement. 200 and 500 μL DPBS were added to the apical and basolateral chambers, respectively. Three measurements spaced at 120o from each other were made for each culture and an average was calculated. Raw data are presented in Supplementary Table 1. 2.6. Cilia beating frequency (CBF) Cultures were equilibrated to 30 °C on a heated stage before the measurement. Videos of cilia motility were recorded using a high-speed digital camera (Ammons Engineering, Mount Morris, MI) connected to a Leica DMI4000B microscope (Leica Microsystems, Buffalo Grove, IL). Four fields were randomly selected and recorded for each culture. CBF was analyzed using the Sisson-Ammons Video Analysis system (SAVA system; Ammons Engineering). 2.7. Mucin ELISA Mucin secretion and expression was analyzed using ELISA. Apical washes were collected by washing the apical surface of the cultures with 100 μL DPBS twice. Dithiothreitol (DTT) was added to the apical washes to a final concentration of 0.025 mM. Cell debris was removed by centrifugation at 600 ×g for 10 min at room temperature. Total cell lysate was prepared in the M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) supplemented with 1× SIGMAFAST™ Protease Inhibitor Cocktail (Sigma-Aldrich). For quantifying mucin secretion and expression, 50 μL of the apical washes and 5 μg of protein 80
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Fig. 2. Characterization of DHA deposition in the exposure module. (A) Deposition of DHA from three solution concentrations, i.e., 0.2, 0.4, and 1.0 M, was quantified using HPLC/MS. The average deposition for each position was calculated from the results of 3 independent exposures. Error bars indicate the SEM of the average deposition (n = 3). Deposited doses of 11.6, 19.3, and 45.8 μg/insert were achieved with 0.2, 0.4, and 1.0 M DHA solution concentrations, respectively. (B) Deposition fraction of DHA from each solution concentration was plotted. Error bars indicate the SEM of the deposition fraction calculated from the results of 3 indpendent exposures. Deposition fractions of 94.9%, 79.8%, and 87.1% were acheived with 0.2, 0.4, and 1.0 M of DHA solutions, respectively.
(Fig. 2A). These deposition doses are equivalent to the surface doses of 35, 58, and 139 μg/cm2, respectively. Deposition of DHA between positions was consistent, with all measurements falling within the 95% confidence interval for these concentrations (Fig. 2A). The deposition fraction of nebulization, which is indicative of the aerosolization efficiency, was estimated to be 94.9%, 79.8% and 87.1% for the 0.2, 0.4, and 1.0 M solutions, respectively (Fig. 2B). Taken together, these results demonstrated that a uniform and reproducible mass deposition of DHA aerosols was achieved using the Cloud System. Additionally, deposition of DHA for each dose was monitored gravimetrically using a QCM incorporated in the A1 position of the exposure module (Table 1). QCM measures mass deposition per unit surface area. For the 0.2 and 0.4 M solutions, the deposition doses estimated from the QCM readings were comparable to those measured using chemical analyses. The estimated deposition dose from the 1.0 M solution (i.e., 53.7 μg/cm2), however, was much lower than that measured by chemical analysis (i.e., 138.7 μg/cm2), suggesting that the mass deposition at this concentration may have exceeded the detection limit of the QCM.
2.10. Statistical analyses Statistical analyses were performed using the SigmaPlot version 11.0 statistics package (Systat Software, San Jose, CA). Data were analyzed using one-way ANOVA, followed by Dunnett's test for identifying significant differences between DPBS-treated vehicle control and DHA-treated groups. 3. Results 3.1. Quantification of DHA aerosol deposition To achieve a range of doses for the subsequent cell exposures, DHA aerosols were generated from three working solutions containing 0.2, 0.4, and 1.0 M DHA. A dose-dependent increase in DHA deposition was observed with increasing concentrations of the working solutions. Three independent exposures were conducted to assess the repeatability and uniformity of the aerosol generation. An average of 11.6, 19.3 and 45.8 μg of DHA was deposited in each position within the exposure module when 0.2, 0.4, and 1.0 M working solutions were aerosolized 81
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Table 1 Measured DHA deposition and QCM reading. [DHA Solution] (M)
Mass Deposition (μg/insert)
Surface Dose (μg/cm2)
Deposition Fraction (%)
QCM (μg/cm2)
0.2 0.4 1.0
11.6 ± 2.2 19.3 ± 2.9 45.8 ± 4.7
35.4 ± 1.3 58.3 ± 1.8 138.7 ± 2.9
94.9 ± 9.3 79.8 ± 8.6 87.1 ± 5.6
35.6 ± 6.2 53.3 ± 5.4 53.7 ± 2.4
Fig. 4. Effects of DHA aerosols on ciliary beating frequency (CBF). Cultures were exposed to DHA aerosols once a week for up to 5 exposures. CBF was measured 4 h (A) and 24 h (B) after one (T1), three (T3), and five (T5) exposures. Data (n = 8) are presented as means ± SEM. ⁎, Ɨ, # p < .05 when compared to the respective vehicle controls on T1, T3, and T5. a, b p < .05 when compared to the medium and high dose groups on T1, respectively.
be < 20% for all doses at the time points analyzed, suggesting that the doses selected for cell exposure are minimally cytotoxic for ALI cultures. TEER measurement was used to evaluate the effect of the DHA aerosols on the paracellular barrier properties of the ALI cultures 24 h after the first and fifth exposures (Fig. 3B). No measurable changes in tissue permeability were observed at either time point, suggesting no loss of tissue barrier functions under the current treatment regimen. Induction of apoptosis was assessed by quantifying the formation of apoptotic bodies after 5 weekly treatments. Representative images of cleaved caspase-3 staining in the vehicle- and high dose-treated groups are shown in Fig. 3C (top panel). No significant difference in the number of apoptotic bodies was observed between the vehicle- and DHA-treated groups (Fig. 3C, bottom panel).
Fig. 3. Cytotoxicity of DHA aerosols in the ALI cultures. Cytotoxicity was evaluated by adenylate kinase (AK) assay (A) and transepithelial electrical resistance (TEER) (B). Cultures were exposed to DHA aerosols once a week for up to 5 exposures. Leakage of AK into the apical and basolateral compartments was analyzed 24 h after one (T1), three (T3), and five (T5) exposures. TEER was measured 24 h after one (T1) and five (T5) exposures. (C) Effects of DHA on apoptosis. Apoptotic bodies were detected using a cleaved caspase-3 antibody by immunohistochemistry. Representative images of the vehicle- and high dosetreated cultures were presented. The number of apoptotic bodies was counted and apoptotic index calculated. Data (n = 4) are presented as means ± SEM.
3.3. Effects of DHA aerosols on cilia beating frequency (CBF) CBF was measured 4 h and 24 h after 1, 3, and 5 weekly exposures. Dose-dependent decreases in CBF were observed 4 h after each exposure (Fig. 4A), with the low dose causing no effect and the median and high doses significantly reducing the CBF. Furthermore, the percent decreases in CBF were greater after 3 and 5 exposures. Similar decrease in CBF by the medium and high doses was also observed 24 h after each treatment (Fig. 4B). However, the percentage of decrease was comparable for all three time-points. No difference in percent active area was observed at all doses following each exposure (data not shown).
3.2. Cytotoxicity of DHA aerosols in the ALI cultures Cytotoxicity of DHA aerosols was evaluated using the non-invasive AK cytotoxicity assay (Fig. 3A). Release of AK from damaged cells was quantified 24 h following 1, 3, and 5 weekly exposures. Percent AK release was found comparable to the vehicle-exposed control and to 82
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Fig. 5. Effects of DHA aerosols on mucin secretion. Cultures were exposed to DHA aerosols once a week for 5 weeks. Secretions of MUC5AC (A) and MUC5B (B) were measured 24 h after one (T1), three (T3), and five (T5) exposures. Data (n = 6 for T1 and T3, n = 4 for T5) are presented as means ± SEM. ⁎, +, # p < .05 when compared to the respective vehicle controls on T1, T3, and T5.
Fig. 6. Recovery of CBF and MUC5AC secretion over time following a single DHA aerosol exposure. Cultures were exposed to 139 μg/cm2 of DHA aerosols. CBF (A) and MUC5AC secretion (B) were measured at different time points after a single exposure. Data (n = 3) were presented as means ± SEM. ⁎, + p < .05 when compared to the respective vehicle controls.
3.4. Effect of DHA aerosols on mucin secretion Secretion of two major airway mucins, MUC5AC and MUC5B, was measured 24 h after 1, 3, and 5 weekly DHA aerosol exposures. Concentration-dependent decrease in MUC5AC secretion was observed after each exposure (Fig. 5A). Furthermore, the level of decrease was independent of the number of treatments. Quantification of MUC5B secretion, on the other hand, revealed a significant induction by the high dose only after the first treatment (Fig. 5B). The increase in MUC5B secretion diminished following the third and fifth exposures. No significant changes were observed in the intracellular expression of both MUC5AC and MUC5B proteins (data not shown).
significantly decreased by the DHA treatment in a dose-dependent manner 24 h after the first exposure. However, such an inhibitory effect diminished after 5 weekly exposures. 3.7. Effect of DHA aerosols on the morphology of ALI cultures The morphology of the ALI cultures was evaluated following 5 weekly exposures to DHA. The overall morphology of the DHA-treated (139 μg/cm2) cultures was similar to the vehicle-treated control cultures based on the assessment of the H&E staining (Fig. 8A). Considering the decrease in MUC5AC secretion by DHA treatment, its effects on goblet cell morphology and density were also investigated. Representative images of the PAS staining from the vehicle- and high dose-treated groups are shown in Fig. 8B (top panel). The size, morphology, and density of the goblet cells were comparable between the vehicle- and DHA-treated groups (Fig. 8B).
3.5. Temporal effects of DHA aerosols on CBF and MUC5AC secretion To assess the reversibility of the tissue responses to DHA aerosol exposures, changes in two non-invasive endpoints, CBF and MUC5AC secretion, in response to treatment with the high dose of DHA were monitored for up to 5 days after the first exposure. Although CBF significantly decreased within 24 h after the treatment, it gradually returned to the baseline level after a 72-h recovery (Fig. 6A). Similarly, the decrease in MUC5AC secretion observed 24 h after the exposure also diminished when the cultures were recovered for longer than 72 h (Fig. 6B). The treatment-associated decrease in CBF and MUC5AC secretion was completely reversed before the next weekly exposure.
4. Discussion The structure and function of the in vitro ALI airway tissue model resemble those of human bronchial epithelium. Its unique air-liquid interface allows conducting in vitro exposures in a manner reflecting how airways are exposed to inhaled toxicants in vivo. These features of the ALI tissue model may contribute to generating toxicity data of relevance for predicting human health risks. So far, we have employed this in vitro tissue model to comprehensively assess the toxicity of cigarette smoke and smoke constituents having known adverse responses in the lung (Cao et al., 2017, 2018; Xiong et al., 2018). Our findings
3.6. Effects of DHA aerosols on MMP secretion The effect of DHA aerosols on the secretion of MMPs was assessed 24 h after the first and fifth exposures. Among the 9 MMPs screened, secretions of MMP-10 (Fig. 7A) and MMP-13 (Fig. 7B) were found to be 83
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Fig. 8. Morphological characterization of DHA-treated ALI cultures following 5 weekly treatments. ALI cultures were treated with DHA aerosols once a week for 5 exposures. Tissues were fixed 24 h after the last exposure. Tissue sections were stained with H&E (A) and PAS (B, upper panel). Representative images of the vehicle- and high dose-treated cultures were presented. Density of the goblet cells was quantified (B, lower panel). Data (n = 4) are presented as means ± SEM.
Fig. 7. Secretion of select MMP proteins in response to DHA aerosol exposures. Cultures were treated with DHA aerosols once a week for 5 exposures. Secretions of MMP-10 (A) and MMP-13 (B) into the basal medium were quantified 24 h after one (T1) and five (T5) exposures. Data (n = 4) are presented as means ± SEM. * p < 0 0.05 when compared to the vehicle controls on T1.
in the previous studies (Lenz et al., 2014). Lenz et al. reported that aerosolization of the vehicle alone does not affect cell viability and activation of IL-8 gene expression (Lenz et al., 2014), indicating that operation of the Cloud System does not induce undesirable stresses to the cells. Similarly, our measurements comparing CBF between the incubator control (i.e., naïve ALI cultures) and the DPBS aerosol-treated vehicle control did not reveal significant differences. These observations support the suitability of the Cloud System for conducting studies using liquid aerosols. DHA aerosol concentrations in the air of the third-generation spraying booths reach 238 mg/m3 air (Scientific Committee on Consumer Safety, 2014). During the tanning session, the peak concentration lasts approximately 6 s. Assuming an average inhalation rate in humans of 20 L/min and that 20% of aerosols reach the tracheobronchial region of the airway (the 2nd generation of the lung), the average surface dose is estimated to be 0.59 μg/cm2 (average surface area from trachea to the 2nd generation of the lung is approximately 161 cm2; unpublished results). The highest dose used in our study (i.e., ~139 μg/cm2), therefore, provides a margin of safety of 235, a safety factor that could account for the less-than-chronic exposure schedule employed in the evaluation (Position paper on Margins of Safety (MOS) in human health risk assessment, 2001). It is worth mentioning that these estimates are rough calculations; advanced computational modeling, such as computational fluid dynamics, is required to derive more accurate estimates. This is beyond the scope of the current study. Transient dose- and time-dependent modulation of several diseaserelated endpoints, including CBF, mucin secretion, and MMP secretion, were induced by the non-cytotoxic doses of DHA tested in our study. Mucociliary clearance (MCC) is a critical defense mechanism of airways that facilitates the removal of inhaled toxicants. The rate of MCC is determined by both CBF and mucus homeostasis (Houtmeyers et al., 1999). In this study, we found that DHA significantly decreased CBF
demonstrated that the major in vivo tissue responses associated with these chemicals could also be found in this in vitro system, supporting its potential application as an in vitro alternative for respiratory toxicology evaluation. In this study, we integrated a liquid aerosol exposure system into the test platform and conducted an evaluation on the potential respiratory toxicity of DHA aerosols. Time-dependent responses of a range of disease-related endpoints were evaluated after up to 5 weekly exposures to DHA aerosols. Compared to the conventional liquid treatment in submerged lung cells, aerosol delivery of chemicals to the air-liquid interface of the ALI cultures offers several advantages. First, exposure at the air-liquid interface represents a more realistic exposure method, mimicking in vivo inhalation exposures (Upadhyay and Palmberg, 2018). Second, deposition of chemicals on the cells can be easily and accurately quantified using analytical chemistry, whereas dosimetry calculation of liquid treatment in submerged cells has always been a challenge where advanced computational modeling is usually required (Hinderliter et al., 2010; Teeguarden et al., 2007; Upadhyay and Palmberg, 2018). Lastly, delivery of aerosols at the air–liquid interface not only improves the interactions between cells and chemicals, but also increases aerosol uptake kinetics (Lenz et al., 2013; Loret et al., 2016). Thus, integration of an advanced liquid aerosol generation and exposure method may increase the human relevance and improve the predictability of the test platform for inhalation toxicology research. The Cloud Liquid Aerosol Exposure System manufactured by Vitrocell has been used for generating and delivering aerosolized nanoparticles and pulmonary medicines (Chortarea et al., 2017, 2015; Lenz et al., 2014). Herein, we further demonstrated reproducible, spatially uniform deposition of DHA aerosols on the apical surface of the ALI cultures, with deposition factors comparable to those observed
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considering the topical application of DHA-containing products. The inhalation toxicity of DHA remains largely unknown, although potential inhalation hazards exist via the use of sunless tanning sprays. Herein, we employed an in vitro test platform to assess a panel of tissue responses induced by DHA in the physical form inhaled by humans in tanning booths. Our findings suggest that DHA causes transient adverse effects on the human ALI airway epithelial tissue model under the current test conditions. Furthermore, findings from this study provide additional evidence supporting the application the ALI airway tissue model as a promising in vitro system for generating toxicity data on aerosolized chemicals.
shortly after the exposure (i.e., 4 h); such inhibitory effects were gradually reversed after a 24 h recovery and were at baseline levels after a 72-h recovery. It is noteworthy that the reduction of CBF at 4 h was greater after the third and fifth exposures, while the extent of reduction at 24 h was comparable for all three sampling times. It has been reported that reactive oxygen species negatively affect the beating frequency of ciliated cells (Tuttle et al., 2009). However, we did not observe significant changes in several markers for oxidative stress, including intracellular glutathione levels, production of reactive oxygen species, protein oxidation, and expression of HMOX-1 in DHA-treated cultures (data not shown). Considering the time-dependent reversibility of CBF reduction and lack of induction of oxidative stress, it is more likely that the decrease in CBF is caused by the physical presence of DHA on the ciliary surface of the ALI cultures. Presumably, as cultures recover over time, DHA is gradually taken up by the cells, allowing ciliated cells to return to their normal beating frequency. Mucus provides a protective barrier against inhaled toxicants and plays a key role in MCC (Rose and Voynow, 2006). The gel-forming mucins, MUC5AC and MUC5B, are the major macromolecule components of airway mucus. Changes in their production and secretion, as well as in their relative proportion, have been reported in patients with COPD (Hauber et al., 2006; Livraghi-Butrico et al., 2016; Rose and Voynow, 2006). Assessment of changes in these two mucins, therefore, may provide useful information indicative of the toxicity of test articles. In this study, we observed consistent reduction of MUC5AC secretion 24 h after each exposure, whereas secretion of MUC5B was increased by the highest dose but only after the first exposure. However, similar to CBF, the secretion of MUC5AC returned to the baseline level within 72h post-treatment. The intracellular production of MUC5AC and MUC5B was not altered by DHA exposure, suggesting that the decrease in MUC5AC secretion is not the result of the downregulation of mucin expression. The sudden increase in MUC5B secretion may be seen as a compensatory mechanism for the decrease in MUC5AC secretion. Following repeated DHA treatment, it is possible that the cells become adapted and no longer need compensation in mucin secretion from MUC5B. Any adaptation that occurred during DHA treatment did not alter the density and morphology of the mucin-secreting goblet cells significantly. It is known that inflammatory mediators modulate mucin homeostasis (Elkington and Friedland, 2006; Greenlee et al., 2007). To explore the possible mechanism accounting for the temporary disruption of mucin homeostasis, we measured the secretion of 40 human cytokines and chemokines after the first and fifth exposures. However, none of the inflammatory mediators showed significant changes in response to DHA exposure (data not shown). A study by Deshmukh et al. reported the involvement of MMP-9 in mediating MUC5AC expression (Deshmukh et al., 2005). Secretion of MMP proteins (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, and MMP-13), therefore, was quantified. Concentrations of two MMPs, MMP-10 and MMP-13, were found to be temporarily decreased after a single DHA treatment; their downregulation, however, was reversed to baseline level after 5 weekly treatments. MMP-10 is known to play a beneficial role in response to acute infection by modulating the pro-inflammatory responses of resident and infiltrating macrophages (McMahan et al., 2016). Downregulation of MMP-10 increases the susceptibility of mice to airway infections (McMahan et al., 2016). MMP-13 is involved in inflammatory responses and tissue remodeling in several lung diseases (Grzela et al., 2016). The downregulation of both MMP proteins may have temporarily compromised the pro-inflammatory responses of the DHA-treated ALI cultures, as well as caused the disturbance of mucin homeostasis. However, the temporary nature and small magnitude of the responses suggest that DHA exerts marginal effects, if any, on MCC in the ALI cultures. Existing information on the toxicity and mutagenicity of DHA reported in different studies is conflicting (Gallagher, 2018; Goldman and Blaney, 1962). Most studies evaluate its adverse effects on skin cells,
Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the Food and Drug Administration or the National Toxicology Program positions or policies. Funding This study was funded by the National Toxicology Program under an Interagency Agreement between FDA and NIEHS (FDA IAG #22417-0502 and NIH IAG #AES12013). Acknowledgments The authors thank Dr. Paul Howard for bringing up the health concerns of DHA to our attention. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.tiv.2019.04.007. References Barnes, P.J., 2008. Immunology of asthma and chronic obstructive pulmonary disease. Nat. Rev. Immunol. 8, 183–192. BéruBé, K., Aufderheide, M., Breheny, D., R, C., Combes, R., Duffin, R., Forbes, B., Gray, A., I, H., M, K., et al., 2009. In Vitro Models of Inhalation Toxicity and Disease. 37. ATLA Alternatives to Laboratory Animals, pp. 89–141. Biondi, P.A., Passerò, E., Soncin, S., Bernardi, C., Chiesa, L.M., 2007. Selective determination of dihydroxyacetone in self-tanning creams by HPLC as Pentafluorobenzyloxime derivative. Chromatographia 65, 65–68. Cao, X., Lin, H., Muskhelishvili, L., Latendresse, J., Richter, P., Heflich, R.H., 2015. Tight junction disruption by cadmium in an in vitro human airway tissue model. Respir. Res. 16, 30–44. Cao, X., Muskhelishvili, L., Latendresse, J., Richter, P., Heflich, R.H., 2017. Evaluating the toxicity of cigarette whole smoke solutions in an air-liquid-Interface human in vitro airway tissue model. Toxicol. Sci. 156, 14–24. Cao, X., Wang, Y., Xiong, R., Muskhelishvili, L., Davis, K., Richter, P.A., Heflich, R.H., 2018. Cigarette whole smoke solutions disturb mucin homeostasis in a human in vitro airway tissue model. Toxicology 409, 119–128. Chortarea, S., Clift, M.J.D., Vanhecke, D., Endes, C., Wick, P., Petri-Fink, A., RothenRutishauser, B., 2015. Repeated exposure to carbon nanotube-based aerosols does not affect the functional properties of a 3D human epithelial airway model. Nanotoxicology 9, 983–993. Chortarea, S., Barosova, H., Clift, M.J.D., Wick, P., Petri-Fink, A., Rothen-Rutishauser, B., 2017. Human asthmatic bronchial cells are more susceptible to subchronic repeated exposures of aerosolized carbon nanotubes at occupationally relevant doses than healthy cells. ACS Nano 11, 7615–7625. Costa, D.L., 2008. Alternative test methods in inhalation toxicology: challenges and opportunities. Exp. Toxicol. Pathol. 60, 105–109. Deshmukh, H.S., Case, L.M., Wesselkamper, S.C., Borchers, M.T., Martin, L.D., Shertzer, H.G., Nadel, J.A., Leikauf, G.D., 2005. Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation. Am. J. Respir. Crit. Care Med. 171, 305–314. Dvorak, A., Tilley, A.E., Shaykhiev, R., Wang, R., Crystal, R.G., 2011. Do airway epithelium air–liquid cultures represent the in vivo airway epithelium transcriptome? Am. J. Respir. Cell Mol. Biol. 44, 465–473. Elkington, P.T.G., Friedland, J.S., 2006. Matrix metalloproteinases in destructive pulmonary pathology. Thorax 61, 259–266. Gallagher, M., 2018. Exposure to dihydroxyacetone in sunless tanning products: understanding the risks. J. Dermatol. Nurses' Assoc. 10, 11–17. Garone, M., Howard, J., Fabrikant, J., 2015. A review of common tanning methods. J.
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Clin. Aesthetic Dermatol. 8, 43–47. Ghorani-Azam, A., Riahi-Zanjani, B., Balali-Mood, M., 2016. Effects of air pollution on human health and practical measures for prevention in Iran. J. Res. Med. Sci. 21, 65–107. Goldman, L., Blaney, D.J., 1962. Dihydroxyacetone: recent clinical investigative studies. Arch. Dermatol. 85, 730–734. Greenlee, K.J., Werb, Z., Kheradmand, F., 2007. Matrix metalloproteinases in lung: multiple, multifarious, and multifaceted. Physiol. Rev. 87, 69–98. Grzela, K., Litwiniuk, M., Zagorska, W., Grzela, T., 2016. Airway remodeling in chronic obstructive pulmonary disease and asthma: the role of matrix Metalloproteinase-9. Arch. Immunol. Ther. Exp. 64, 47–55. Hauber, H.-P., Foley, S.C., Hamid, Q., 2006. Mucin overproduction in chronic inflammatory lung disease. Can. Respir. J. 13, 327–335. Hinderliter, P.M., Minard, K.R., Orr, G., Chrisler, W.B., Thrall, B.D., Pounds, J.G., Teeguarden, J.G., 2010. ISDD: a computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies. Particle Fibre Toxicol. 7, 36–55. Houtmeyers, E., Gosselink, R., Gayan-Ramirez, G., Decramer, M., 1999. Regulation of mucociliary clearance in health and disease. Eur. Respir. J. 13, 1177–1188. Kesimer, M., Kirkham, S., Pickles, R.J., Henderson, A.G., Alexis, N.E., DeMaria, G., Knight, D., Thornton, D.J., Sheehan, J.K., 2009. Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways? Am. J. Phys. Lung Cell. Mol. Phys. 296, L92–L100. Knight, D.A., Holgate, S.T., 2003. The airway epithelium: structural and functional properties in health and disease. Respirology 8, 432–446. Lenz, A.-G., Karg, E., Brendel, E., Hinze-Heyn, H., Maier, K.L., Eickelberg, O., Stoeger, T., Schmid, O., 2013. Inflammatory and oxidative stress responses of an alveolar epithelial cell line to airborne zinc oxide nanoparticles at the air-liquid Interface: a comparison with conventional, submerged cell-culture conditions. Biomed. Res. Int. 2013, 652632. Lenz, A.-G., Stoeger, T., Cei, D., Schmidmeir, M., Semren, N., Burgstaller, G., Lentner, B., Eickelberg, O., Meiners, S., Schmid, O., 2014. Efficient bioactive delivery of aerosolized drugs to human pulmonary epithelial cells cultured in air–liquid Interface conditions. Am. J. Respir. Cell Mol. Biol. 51, 526–535. Levy, S.B., 1992. Dihydroxyacetone-containing sunless or self-tanning lotions. J. Am. Acad. Dermatol. 27, 989–993. Livraghi-Butrico, A., Grubb, B.R., Wilkinson, K.J., Volmer, A.S., Burns, K.A., Evans, C.M., O'Neal, W.K., Boucher, R.C., 2016. Contribution of mucus concentration and secreted mucins Muc5ac and Muc5b to the pathogenesis of muco-obstructive lung disease. Mucosal Immunol. 10, 395–407. Loret, T., Peyret, E., Dubreuil, M., Aguerre-Chariol, O., Bressot, C., le Bihan, O., Amodeo,
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Assessing the respiratory toxicity of dihydroxyacetone using an in vitro human airway epithelial tissue model Yiying Wanga, Qiangen Wub, Levan Muskhelishvilic, Kelly Davisc, Matthew Bryantb, Xuefei Caoa,
T ⁎
a
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States of America Division of Biochemistry Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States of America c Toxicologic Pathology Associates, Jefferson, AR 72079, United States of America b
A R T I C LE I N FO
A B S T R A C T
Keywords: In vitro human air-liquid-interface (ALI) airway epithelial tissue model Dihydroxyacetone (DHA) Respiratory toxicity Cloud liquid aerosol generation and exposure system
Dihydroxyacetone (DHA) is an approved color additive used in sunless tanning lotions. Recently, there has been an increased use of DHA in sunless tanning booths in a manner that could result in its inhalation during application. In the present study, we have evaluated the potential for DHA causing toxicity via inhalation using a human air-liquid-interface (ALI) in vitro airway epithelial tissue model. ALI airway models have a close structural and functional resemblance to the in vivo airway epithelium, and thus data generated in these models may have relevance for predicting human responses. To simulate in vivo exposure conditions, we employed a method for liquid aerosol generation that mimics the physical form of inhaled chemicals and used doses of DHA and an exposure frequency reflecting human respiratory exposures during tanning sessions. Compared to the vehicle control, cilia beating frequency (CBF) and MUC5AC secretion were significantly decreased after each exposure. However, time-course studies indicated that both CBF and MUC5AC secretion returned to normal levels within 3 days after the treatment. Matrix metalloproteinase (MMP) release, on the other hand, was decreased 24 h after the first exposure and its level returned to baseline after 5 exposures. No significant morphological changes occurred in the DHA-treated cultures after 5 weekly exposures. Our findings indicate that DHA, at concentrations likely to be experienced by humans, has transient toxic effects on human airway ALI cultures.
1. Introduction Inhalation exposures to toxicants are associated with asthma, chronic obstructive pulmonary disease (COPD), and lung cancer (Barnes, 2008; BéruBé et al., 2009; Ghorani-Azam et al., 2016; Knight and Holgate, 2003; Pope et al., 2002). The lung serves both as a target tissue and portal of entry for toxicants into the systemic circulation. Characterizing adverse responses in airway epithelium, therefore, could provide valuable information for evaluating health hazards from inhalation exposures. Traditionally, rodent models are considered the gold standard for inhalation toxicology studies. However, critical differences in dosimetry, geometry, and physiology between rodent and human respiratory systems raise concerns over extrapolating human responses from rodent test data. Furthermore, the cost and time involved in conducting in vivo inhalation testing make it challenging to obtain inhalation toxicity data on the large number of new and existing chemicals, respiratory toxicants, and inhaled therapeutics (Costa, 2008). Together with the increasing pressure from animal rights groups, it is desirable to develop alternative human-based in vitro
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approaches for inhalation toxicity screening and testing. A number of in vitro lung models are available and have been used for inhalation studies (BéruBé et al., 2009). Among them, the human in vitro air-liquid-interface (ALI) airway epithelial tissue model has received increasing attention for its potential applications in inhalation toxicology (BéruBé et al., 2009; Cao et al., 2015, 2017; Chortarea et al., 2015). The well-differentiated ALI airway tissue model is derived from primary human tracheobronchial epithelial cells cultured at the air-liquid interface. Unlike conventional monolayer cell lines generated from a single cell type, the ALI tissue model consists of three major airway epithelial cell types, i.e., ciliated cells, goblet cells, and basal cells, arranged in a pseudostratified structure. Key in vivo functions, such as tight junctions and trans-epithelial electrical resistance, are retained (Cao et al., 2015, 2017). Genetic and proteomic analyses have further revealed a strong resemblance between the transcriptome and secretome of ALI models and human airway epithelium (Dvorak et al., 2011; Kesimer et al., 2009). Besides the structural and functional similarities, the presence of the unique air-liquid interface of the ALI cultures makes direct exposures to aerosols, vapors, and particles possible. Thus, the
Corresponding author at: U.S. FDA/NCTR, Division of Genetic and Molecular Toxicology, 3900 NCTR Road, Jefferson, AR 72079, USA. E-mail address: [email protected] (X. Cao).
https://doi.org/10.1016/j.tiv.2019.04.007 Received 15 February 2019; Received in revised form 3 April 2019; Accepted 4 April 2019 Available online 05 April 2019 0887-2333/ Published by Elsevier Ltd.
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cells at 37 °C during the exposures (Fig. 1). The operational principal of the Cloud System has been described by Lenz et al. (Lenz et al., 2014). 1,3-Dihydroxyacetone (DHA, Toronto Research Chemicals, North York, Canada) was dissolved in diluted Dulbecco's Phosphate-Buffered Saline (DPBS, pH 7.4, Corning, Manassas, VA; 1:80 diluted in molecular grade water) to concentrations of 0.2, 0.4, and 1.0 M. To quantify aerosol deposition, 24-well PET Transwell® cell culture inserts (Corning, Tewksbury, MA) containing 100 μL DPBS were placed in all eight positions of the exposure module pre-warmed to 37 °C. 1 mL DHA solution was added to the nebulizer and immediately nebulized for 30 s. The exposure chamber was removed 20 min after the nebulization to ensure complete settlement of the aerosols onto the culture inserts. 90 μL DPBS containing the deposited DHA were collected from each insert for chemistry analysis. DHA was quantified using a method described by Biondi et al. (2007) with a few modifications. Briefly, 10 μL of standards or samples were derivatized for 30 min at 65 °C in 5 mg/mL O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride (PFBHA, Sigma-Aldrich, St. Louis, MO) prepared in 10 mM ammonium acetate solution (pH 4.0). The reaction mixture was cooled down on ice and diluted in 4 volumes of acetonitrile. 10 μL dilutions were injected into an HPLC equipped with a Waters 2695 separation module connected to an Atlantis T3 column (4.6 mm × 150 mm, 5 μm) and a Waters ACQUITY QDa Mass Detector. The derivatization product was eluted in an isocratic elution using a mobile phase composed of water (Mobile A) and HPLC-grade acetonitrile (Mobile B), both spiked with 0.1% formic acid. Elution was conducted with 80% of Mobile B at a flow rate of 0.5 mL/min for 5 min. The temperature of the HPLC column was set at 40 °C. Elution of DHA was monitored at m/z 286.0. The amount of DHA was calculated using a linear standard curve with a concentration range from 1.6 to 1000 μg/ mL (R2 = 0.99). The deposition factors for DHA aerosols were calculated using the following formula: deposition factor = (DHA deposition)measured/(DHA deposition)target × 100%, where measured DHA deposition was the mass of DHA quantified in each insert using chemical analysis and target DHA deposition was defined as ([DHA]working solution × nebulization volume) × (surface area of each insert/surface area of the exposure module).
toxicity of inhaled toxicants can be evaluated in these tissue models using bioassays reflecting tissue responses and under conditions mimicking in vivo inhalation exposures. DHA is an approved color additive used in sunless tanning lotions (U.S. Food and Drug Administration, 2016). Its glycosidic hydroxyl group reacts with amino groups of epidermal proteins via non-enzymatic glycation, giving the skin a brownish color as the result of the formation of melanoidins (Gallagher, 2018; Mullarkey et al., 1990; Namiki, 1988). DHA use is restricted to external applications; because relevant safety data are lacking, its contact with body surface covered by mucous membranes is strongly discouraged (Office of Cosmetics and Colors et al., 2003; Scientific Committee on Consumer Safety, 2014; U.S. Food and Drug Administration, 2016). However, unintentional exposure of respiratory mucosal surfaces to DHA aerosols is highly possible during sunless tanning sessions, a possibility for which it is not approved (Scientific Committee on Consumer Safety, 2014). In this study, we evaluated the potential respiratory toxicity of DHA using the ALI airway tissue model. To mimic in vivo exposure conditions, we used a liquid aerosol generation and exposure system (Chortarea et al., 2015; Lenz et al., 2014) and exposed ALI cultures to a range of non-cytotoxic doses of DHA aerosols at the air-liquid interface. A weekly treatment regimen was employed to mimic the tanning frequency of consumers. ALI cultures were repeatedly exposed to DHA aerosols once per week for up to 5 weeks. A panel of endpoints related to tissue responses was assessed at several time points after each exposure to understand the temporal response and recovery of the ALI cultures. 2. Materials and methods 2.1. Quantification of DHA aerosol deposition The design of the Vitrocell® Cloud 12/12 Liquid Aerosol Exposure System (Waldkirch, Germany) is schematically illustrated in Fig. 1. The Cloud System consists of a nebulizer with a perforated, piezoelectrically driven vibrating membrane (Aeroneb® Pro, Aerogen, Galway, Ireland), a removable exposure chamber for confining the aerosols, an exposure module with an integrated quartz crystal microbalance (QCM) for realtime monitoring of mass deposition, and a heating unit for maintaining
2.2. Human ALI airway tissue model Human ALI airway tissue models were established using the PneumaCult™-ALI Medium kit (STEMCELL Technologies, Vancouver, Canada) as described previously (Cao et al., 2017). Briefly, normal human primary tracheobronchial epithelial cells (The CF Center Tissue Procurement and Cell Culture Core, University of North Carolina, Chapel Hill, NC) were expanded on 100-mm tissue culture dishes in PneumaCult™-Ex Expansion Medium (STEMCELL Technologies) until cells reached approximately 80% confluence. Cells were collected by trypsinization and resuspended in the Expansion Medium at a density of 4 × 105 cells/mL; 100 μL cell suspensions were added to each 24-well PET Transwell® cell culture insert. Expansion medium was added to both the apical and basolateral compartments to support cell proliferation. When cells grew to 100% confluence, the medium in the apical compartment was removed and cells were fed from the basolateral side only with PneumaCult™-ALI Maintenance Medium every other day for 4 weeks until they develop the structure of pseudostratified ciliated epithelium. Fig. 1. Schematic diagram of the VITROCELL® Cloud 12 Liquid Aerosol Exposure System. The Cloud 12 consists of an exposure chamber, an exposure module, and an electronically controlled heating unit. A Quartz Crystal Microbalance (QCM) Sensor is installed at the A1 position of the exposure module for real-time dose monitoring. The exposure chamber is divided into two unequal compartments, each equipped with a vibrating mesh nebulizer on the top. Such a setup allows concurrent exposures to the vehicles and test articles.
2.3. DHA aerosol exposure Dulbecco's Modified Eagle's Medium (DMEM, American Type Culture Collection, Manassas, VA), supplemented with 27 mM 4-(2hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) and 0.1 mM sodium pyruvate (Life Technologies, Grand Island, NY), was used as the exposure medium. 3.5 mL of pre-warmed media were added to each 79
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lysates were coated on a high-protein-binding ELISA plate (Thermo Fisher Scientific). The ELISA plates were first blocked with bovine serum albumin (BSA; 10 mg/mL) for 1 h at room temperature, followed by a 1-h incubation with anti-MUC5AC (45 M1) (Pierce) or anti-MUC5B antibodies (Abcam, Cambridge, MA) (1:500 dilution in DPBS containing 10 mg/mL BSA, 0.3% Triton X-100, and 0.2% Tween-20). 100 μL HRPconjugated goat-anti-mouse antibody (1:1000 dilution in DPBS containing 10 mg/mL BSA, 0.3% Triton X-100, and 0.2% Tween-20; Rockland, Limerick, PA) then were added to each well and incubated for 30 min. Excess secondary antibodies were removed by washing with DPBS three times. Color was developed by adding 50 μL of TMB substrate (Thermo Fisher Scientific) to each well; color reactions were stopped with 50 μL of 2 N HCl. The absorbance at 450 nm was measured using a Synergy H4 microplate reader. Raw data are presented in Supplementary Tables 2 to 4.
well in the pre-warmed exposure module; culture inserts were hung in each well by the plastic rim. The basolateral side of the inserts touched the exposure medium and the apical side was exposed to air for aerosol exposures. DHA aerosols were generated as described in Quantification of DHA aerosol deposition. Upon conclusion of the exposure, only the basolateral sides of the cultures were washed with DPBS, leaving the apical surface undisturbed; 400 μL fresh Maintenance Media were added to the basolateral compartment. The cultures were maintained in a humidified incubator at 37 °C with 5% CO2. By handling cultures this way, cells were continuously exposed to DHA aerosols from the apical side until the first mucus collection that occurred at 24 h after the exposure. Spray tan from one tanning session usually lasts for 5 to 7 days (Garone et al., 2015; Levy, 1992). Spray tanning, therefore, typically is conducted every 7 days to maintain the color. To mimic the tanning frequency by consumers, ALI cultures were exposed to DHA aerosols once per week for up to 5 weeks.
2.8. Matrix metalloproteinase (MMP) Bio-plex assay
2.4. Adenylate kinase (AK) cytotoxicity assay
Secretion of select MMP proteins into the basolateral media was quantified using the Bio-plex Pro Human MMP 9-plex assay kit and a MAGPIX system (Bio-Rad, Hercules, CA). 50 μL basolateral media collected 24 h after the first and fifth exposures were assayed. The assays were performed by following the manufacturer's instructions. Concentrations of the MMP proteins were calculated using a concurrent standard and Bio-Rad Bio-Plex Manager software (version 4.1). Raw data are presented in Supplementary Table 5.
The cytotoxicity of DHA aerosols was assessed by measuring the release of intracellular Adenylate Kinase (AK) from damaged cells using a ToxiLight™ Non-destructive Cytotoxicity BioAssay Kit (Lonza, Walkersville, MD). 24 h after the exposure, the apical side of the cultures was washed twice with 100 μL DPBS and the apical washes were combined into a microcentrifuge tube; basolateral medium was collected into a separate microcentrifuge tube. For the AK cytotoxicity assay, 70 μL basolateral media and 35 μL apical washes were combined and incubated with the AK Detection Reagent for 5 min. Luminescence was measured using a Synergy H4 microplate reader (BioTek, Winooski, VT). Total AK release was determined by assaying basolateral medium from cultures treated with 1% Triton™ X-100 for 24 h at 37 °C. Results are presented as percent AK release.
2.9. Histology The ALI cultures were washed briefly with DPBS and fixed in 10% neutral buffered formalin for 48 h. The membranes were routinely processed and embedded in paraffin. 5-μm thick tissue sections were cut, mounted on positively charged slides, and deparaffinized and hydrated by processing through xylene and graded ethanol solutions. Hematoxylin and eosin (H&E) staining was conducted using a Leica Autostainer. For evaluation of the apoptotic index, deparaffinized tissue sections were immunohistochemically stained with a cleaved caspase-3 antibody. Briefly, the cleaved caspase-3 antigen was retrieved by boiling the tissue sections in 0.01 M citrate buffer, pH 6.0, for 15 min (3 × 5 min) using a microwave oven. The slides were washed in H2O for 5 min after cooling to room temperature. Endogenous peroxidase was then quenched by treating tissue sections with 3% hydrogen peroxide containing 0.1% sodium azide for 10 min. After blocking the nonspecific binding in 0.5% casein for 20 min, the slides were incubated for 1 h with the rabbit polyclonal cleaved caspase-3 antibody (1:200 dilution in DPBS containing 1% BSA; Cell Signaling, Danvers, MA), followed by sequential incubation with biotinylated goat-antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and ExtrAvidin-conjugated horseradish peroxidase (Sigma-Aldrich) at dilutions of 1:200 and 1:100, respectively. Staining was developed with diaminobenzidine substrate (DAB; Sigma-Aldrich) for 5 min. Sections were then counter-stained with hematoxylin and mounted with Permount™ Mounting Medium (Thermo Fisher Scientific). Rabbit IgG was used in place of primary antibody to serve as the negative control. For evaluating goblet cell hyperplasia/hypoplasia, a separate set of sections was histochemically stained with the periodic acid Schiff (PAS) reaction. PAS staining labels glycoprotein and glycolipid components in mucins. It is widely used for identifying mucus-secreting goblet cells and assessing the morphology of goblet cells. The slides were incubated in periodic acid solution for 5 min at room temperature. The slides were then washed with tap water and incubated in Schiff's reagent for 30 min at room temperature, followed by additional washing in running tap water. Sections were counter-stained with hematoxylin and coverslipped with Permount™ Mounting Medium.
2.5. Trans-epithelial electrical resistance (TEER) TEER was measured using an EVOM2 epithelial volt-ohmmeter and an STX2 electrode (World Precision Instruments, Sarasota, FL). Changes in TEER values indicate the effect of treatments on paracellular barrier properties. The EVOM2 was calibrated using a test resistor prior to the measurement. 200 and 500 μL DPBS were added to the apical and basolateral chambers, respectively. Three measurements spaced at 120o from each other were made for each culture and an average was calculated. Raw data are presented in Supplementary Table 1. 2.6. Cilia beating frequency (CBF) Cultures were equilibrated to 30 °C on a heated stage before the measurement. Videos of cilia motility were recorded using a high-speed digital camera (Ammons Engineering, Mount Morris, MI) connected to a Leica DMI4000B microscope (Leica Microsystems, Buffalo Grove, IL). Four fields were randomly selected and recorded for each culture. CBF was analyzed using the Sisson-Ammons Video Analysis system (SAVA system; Ammons Engineering). 2.7. Mucin ELISA Mucin secretion and expression was analyzed using ELISA. Apical washes were collected by washing the apical surface of the cultures with 100 μL DPBS twice. Dithiothreitol (DTT) was added to the apical washes to a final concentration of 0.025 mM. Cell debris was removed by centrifugation at 600 ×g for 10 min at room temperature. Total cell lysate was prepared in the M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) supplemented with 1× SIGMAFAST™ Protease Inhibitor Cocktail (Sigma-Aldrich). For quantifying mucin secretion and expression, 50 μL of the apical washes and 5 μg of protein 80
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Fig. 2. Characterization of DHA deposition in the exposure module. (A) Deposition of DHA from three solution concentrations, i.e., 0.2, 0.4, and 1.0 M, was quantified using HPLC/MS. The average deposition for each position was calculated from the results of 3 independent exposures. Error bars indicate the SEM of the average deposition (n = 3). Deposited doses of 11.6, 19.3, and 45.8 μg/insert were achieved with 0.2, 0.4, and 1.0 M DHA solution concentrations, respectively. (B) Deposition fraction of DHA from each solution concentration was plotted. Error bars indicate the SEM of the deposition fraction calculated from the results of 3 indpendent exposures. Deposition fractions of 94.9%, 79.8%, and 87.1% were acheived with 0.2, 0.4, and 1.0 M of DHA solutions, respectively.
(Fig. 2A). These deposition doses are equivalent to the surface doses of 35, 58, and 139 μg/cm2, respectively. Deposition of DHA between positions was consistent, with all measurements falling within the 95% confidence interval for these concentrations (Fig. 2A). The deposition fraction of nebulization, which is indicative of the aerosolization efficiency, was estimated to be 94.9%, 79.8% and 87.1% for the 0.2, 0.4, and 1.0 M solutions, respectively (Fig. 2B). Taken together, these results demonstrated that a uniform and reproducible mass deposition of DHA aerosols was achieved using the Cloud System. Additionally, deposition of DHA for each dose was monitored gravimetrically using a QCM incorporated in the A1 position of the exposure module (Table 1). QCM measures mass deposition per unit surface area. For the 0.2 and 0.4 M solutions, the deposition doses estimated from the QCM readings were comparable to those measured using chemical analyses. The estimated deposition dose from the 1.0 M solution (i.e., 53.7 μg/cm2), however, was much lower than that measured by chemical analysis (i.e., 138.7 μg/cm2), suggesting that the mass deposition at this concentration may have exceeded the detection limit of the QCM.
2.10. Statistical analyses Statistical analyses were performed using the SigmaPlot version 11.0 statistics package (Systat Software, San Jose, CA). Data were analyzed using one-way ANOVA, followed by Dunnett's test for identifying significant differences between DPBS-treated vehicle control and DHA-treated groups. 3. Results 3.1. Quantification of DHA aerosol deposition To achieve a range of doses for the subsequent cell exposures, DHA aerosols were generated from three working solutions containing 0.2, 0.4, and 1.0 M DHA. A dose-dependent increase in DHA deposition was observed with increasing concentrations of the working solutions. Three independent exposures were conducted to assess the repeatability and uniformity of the aerosol generation. An average of 11.6, 19.3 and 45.8 μg of DHA was deposited in each position within the exposure module when 0.2, 0.4, and 1.0 M working solutions were aerosolized 81
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Table 1 Measured DHA deposition and QCM reading. [DHA Solution] (M)
Mass Deposition (μg/insert)
Surface Dose (μg/cm2)
Deposition Fraction (%)
QCM (μg/cm2)
0.2 0.4 1.0
11.6 ± 2.2 19.3 ± 2.9 45.8 ± 4.7
35.4 ± 1.3 58.3 ± 1.8 138.7 ± 2.9
94.9 ± 9.3 79.8 ± 8.6 87.1 ± 5.6
35.6 ± 6.2 53.3 ± 5.4 53.7 ± 2.4
Fig. 4. Effects of DHA aerosols on ciliary beating frequency (CBF). Cultures were exposed to DHA aerosols once a week for up to 5 exposures. CBF was measured 4 h (A) and 24 h (B) after one (T1), three (T3), and five (T5) exposures. Data (n = 8) are presented as means ± SEM. ⁎, Ɨ, # p < .05 when compared to the respective vehicle controls on T1, T3, and T5. a, b p < .05 when compared to the medium and high dose groups on T1, respectively.
be < 20% for all doses at the time points analyzed, suggesting that the doses selected for cell exposure are minimally cytotoxic for ALI cultures. TEER measurement was used to evaluate the effect of the DHA aerosols on the paracellular barrier properties of the ALI cultures 24 h after the first and fifth exposures (Fig. 3B). No measurable changes in tissue permeability were observed at either time point, suggesting no loss of tissue barrier functions under the current treatment regimen. Induction of apoptosis was assessed by quantifying the formation of apoptotic bodies after 5 weekly treatments. Representative images of cleaved caspase-3 staining in the vehicle- and high dose-treated groups are shown in Fig. 3C (top panel). No significant difference in the number of apoptotic bodies was observed between the vehicle- and DHA-treated groups (Fig. 3C, bottom panel).
Fig. 3. Cytotoxicity of DHA aerosols in the ALI cultures. Cytotoxicity was evaluated by adenylate kinase (AK) assay (A) and transepithelial electrical resistance (TEER) (B). Cultures were exposed to DHA aerosols once a week for up to 5 exposures. Leakage of AK into the apical and basolateral compartments was analyzed 24 h after one (T1), three (T3), and five (T5) exposures. TEER was measured 24 h after one (T1) and five (T5) exposures. (C) Effects of DHA on apoptosis. Apoptotic bodies were detected using a cleaved caspase-3 antibody by immunohistochemistry. Representative images of the vehicle- and high dosetreated cultures were presented. The number of apoptotic bodies was counted and apoptotic index calculated. Data (n = 4) are presented as means ± SEM.
3.3. Effects of DHA aerosols on cilia beating frequency (CBF) CBF was measured 4 h and 24 h after 1, 3, and 5 weekly exposures. Dose-dependent decreases in CBF were observed 4 h after each exposure (Fig. 4A), with the low dose causing no effect and the median and high doses significantly reducing the CBF. Furthermore, the percent decreases in CBF were greater after 3 and 5 exposures. Similar decrease in CBF by the medium and high doses was also observed 24 h after each treatment (Fig. 4B). However, the percentage of decrease was comparable for all three time-points. No difference in percent active area was observed at all doses following each exposure (data not shown).
3.2. Cytotoxicity of DHA aerosols in the ALI cultures Cytotoxicity of DHA aerosols was evaluated using the non-invasive AK cytotoxicity assay (Fig. 3A). Release of AK from damaged cells was quantified 24 h following 1, 3, and 5 weekly exposures. Percent AK release was found comparable to the vehicle-exposed control and to 82
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Fig. 5. Effects of DHA aerosols on mucin secretion. Cultures were exposed to DHA aerosols once a week for 5 weeks. Secretions of MUC5AC (A) and MUC5B (B) were measured 24 h after one (T1), three (T3), and five (T5) exposures. Data (n = 6 for T1 and T3, n = 4 for T5) are presented as means ± SEM. ⁎, +, # p < .05 when compared to the respective vehicle controls on T1, T3, and T5.
Fig. 6. Recovery of CBF and MUC5AC secretion over time following a single DHA aerosol exposure. Cultures were exposed to 139 μg/cm2 of DHA aerosols. CBF (A) and MUC5AC secretion (B) were measured at different time points after a single exposure. Data (n = 3) were presented as means ± SEM. ⁎, + p < .05 when compared to the respective vehicle controls.
3.4. Effect of DHA aerosols on mucin secretion Secretion of two major airway mucins, MUC5AC and MUC5B, was measured 24 h after 1, 3, and 5 weekly DHA aerosol exposures. Concentration-dependent decrease in MUC5AC secretion was observed after each exposure (Fig. 5A). Furthermore, the level of decrease was independent of the number of treatments. Quantification of MUC5B secretion, on the other hand, revealed a significant induction by the high dose only after the first treatment (Fig. 5B). The increase in MUC5B secretion diminished following the third and fifth exposures. No significant changes were observed in the intracellular expression of both MUC5AC and MUC5B proteins (data not shown).
significantly decreased by the DHA treatment in a dose-dependent manner 24 h after the first exposure. However, such an inhibitory effect diminished after 5 weekly exposures. 3.7. Effect of DHA aerosols on the morphology of ALI cultures The morphology of the ALI cultures was evaluated following 5 weekly exposures to DHA. The overall morphology of the DHA-treated (139 μg/cm2) cultures was similar to the vehicle-treated control cultures based on the assessment of the H&E staining (Fig. 8A). Considering the decrease in MUC5AC secretion by DHA treatment, its effects on goblet cell morphology and density were also investigated. Representative images of the PAS staining from the vehicle- and high dose-treated groups are shown in Fig. 8B (top panel). The size, morphology, and density of the goblet cells were comparable between the vehicle- and DHA-treated groups (Fig. 8B).
3.5. Temporal effects of DHA aerosols on CBF and MUC5AC secretion To assess the reversibility of the tissue responses to DHA aerosol exposures, changes in two non-invasive endpoints, CBF and MUC5AC secretion, in response to treatment with the high dose of DHA were monitored for up to 5 days after the first exposure. Although CBF significantly decreased within 24 h after the treatment, it gradually returned to the baseline level after a 72-h recovery (Fig. 6A). Similarly, the decrease in MUC5AC secretion observed 24 h after the exposure also diminished when the cultures were recovered for longer than 72 h (Fig. 6B). The treatment-associated decrease in CBF and MUC5AC secretion was completely reversed before the next weekly exposure.
4. Discussion The structure and function of the in vitro ALI airway tissue model resemble those of human bronchial epithelium. Its unique air-liquid interface allows conducting in vitro exposures in a manner reflecting how airways are exposed to inhaled toxicants in vivo. These features of the ALI tissue model may contribute to generating toxicity data of relevance for predicting human health risks. So far, we have employed this in vitro tissue model to comprehensively assess the toxicity of cigarette smoke and smoke constituents having known adverse responses in the lung (Cao et al., 2017, 2018; Xiong et al., 2018). Our findings
3.6. Effects of DHA aerosols on MMP secretion The effect of DHA aerosols on the secretion of MMPs was assessed 24 h after the first and fifth exposures. Among the 9 MMPs screened, secretions of MMP-10 (Fig. 7A) and MMP-13 (Fig. 7B) were found to be 83
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Fig. 8. Morphological characterization of DHA-treated ALI cultures following 5 weekly treatments. ALI cultures were treated with DHA aerosols once a week for 5 exposures. Tissues were fixed 24 h after the last exposure. Tissue sections were stained with H&E (A) and PAS (B, upper panel). Representative images of the vehicle- and high dose-treated cultures were presented. Density of the goblet cells was quantified (B, lower panel). Data (n = 4) are presented as means ± SEM.
Fig. 7. Secretion of select MMP proteins in response to DHA aerosol exposures. Cultures were treated with DHA aerosols once a week for 5 exposures. Secretions of MMP-10 (A) and MMP-13 (B) into the basal medium were quantified 24 h after one (T1) and five (T5) exposures. Data (n = 4) are presented as means ± SEM. * p < 0 0.05 when compared to the vehicle controls on T1.
in the previous studies (Lenz et al., 2014). Lenz et al. reported that aerosolization of the vehicle alone does not affect cell viability and activation of IL-8 gene expression (Lenz et al., 2014), indicating that operation of the Cloud System does not induce undesirable stresses to the cells. Similarly, our measurements comparing CBF between the incubator control (i.e., naïve ALI cultures) and the DPBS aerosol-treated vehicle control did not reveal significant differences. These observations support the suitability of the Cloud System for conducting studies using liquid aerosols. DHA aerosol concentrations in the air of the third-generation spraying booths reach 238 mg/m3 air (Scientific Committee on Consumer Safety, 2014). During the tanning session, the peak concentration lasts approximately 6 s. Assuming an average inhalation rate in humans of 20 L/min and that 20% of aerosols reach the tracheobronchial region of the airway (the 2nd generation of the lung), the average surface dose is estimated to be 0.59 μg/cm2 (average surface area from trachea to the 2nd generation of the lung is approximately 161 cm2; unpublished results). The highest dose used in our study (i.e., ~139 μg/cm2), therefore, provides a margin of safety of 235, a safety factor that could account for the less-than-chronic exposure schedule employed in the evaluation (Position paper on Margins of Safety (MOS) in human health risk assessment, 2001). It is worth mentioning that these estimates are rough calculations; advanced computational modeling, such as computational fluid dynamics, is required to derive more accurate estimates. This is beyond the scope of the current study. Transient dose- and time-dependent modulation of several diseaserelated endpoints, including CBF, mucin secretion, and MMP secretion, were induced by the non-cytotoxic doses of DHA tested in our study. Mucociliary clearance (MCC) is a critical defense mechanism of airways that facilitates the removal of inhaled toxicants. The rate of MCC is determined by both CBF and mucus homeostasis (Houtmeyers et al., 1999). In this study, we found that DHA significantly decreased CBF
demonstrated that the major in vivo tissue responses associated with these chemicals could also be found in this in vitro system, supporting its potential application as an in vitro alternative for respiratory toxicology evaluation. In this study, we integrated a liquid aerosol exposure system into the test platform and conducted an evaluation on the potential respiratory toxicity of DHA aerosols. Time-dependent responses of a range of disease-related endpoints were evaluated after up to 5 weekly exposures to DHA aerosols. Compared to the conventional liquid treatment in submerged lung cells, aerosol delivery of chemicals to the air-liquid interface of the ALI cultures offers several advantages. First, exposure at the air-liquid interface represents a more realistic exposure method, mimicking in vivo inhalation exposures (Upadhyay and Palmberg, 2018). Second, deposition of chemicals on the cells can be easily and accurately quantified using analytical chemistry, whereas dosimetry calculation of liquid treatment in submerged cells has always been a challenge where advanced computational modeling is usually required (Hinderliter et al., 2010; Teeguarden et al., 2007; Upadhyay and Palmberg, 2018). Lastly, delivery of aerosols at the air–liquid interface not only improves the interactions between cells and chemicals, but also increases aerosol uptake kinetics (Lenz et al., 2013; Loret et al., 2016). Thus, integration of an advanced liquid aerosol generation and exposure method may increase the human relevance and improve the predictability of the test platform for inhalation toxicology research. The Cloud Liquid Aerosol Exposure System manufactured by Vitrocell has been used for generating and delivering aerosolized nanoparticles and pulmonary medicines (Chortarea et al., 2017, 2015; Lenz et al., 2014). Herein, we further demonstrated reproducible, spatially uniform deposition of DHA aerosols on the apical surface of the ALI cultures, with deposition factors comparable to those observed
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considering the topical application of DHA-containing products. The inhalation toxicity of DHA remains largely unknown, although potential inhalation hazards exist via the use of sunless tanning sprays. Herein, we employed an in vitro test platform to assess a panel of tissue responses induced by DHA in the physical form inhaled by humans in tanning booths. Our findings suggest that DHA causes transient adverse effects on the human ALI airway epithelial tissue model under the current test conditions. Furthermore, findings from this study provide additional evidence supporting the application the ALI airway tissue model as a promising in vitro system for generating toxicity data on aerosolized chemicals.
shortly after the exposure (i.e., 4 h); such inhibitory effects were gradually reversed after a 24 h recovery and were at baseline levels after a 72-h recovery. It is noteworthy that the reduction of CBF at 4 h was greater after the third and fifth exposures, while the extent of reduction at 24 h was comparable for all three sampling times. It has been reported that reactive oxygen species negatively affect the beating frequency of ciliated cells (Tuttle et al., 2009). However, we did not observe significant changes in several markers for oxidative stress, including intracellular glutathione levels, production of reactive oxygen species, protein oxidation, and expression of HMOX-1 in DHA-treated cultures (data not shown). Considering the time-dependent reversibility of CBF reduction and lack of induction of oxidative stress, it is more likely that the decrease in CBF is caused by the physical presence of DHA on the ciliary surface of the ALI cultures. Presumably, as cultures recover over time, DHA is gradually taken up by the cells, allowing ciliated cells to return to their normal beating frequency. Mucus provides a protective barrier against inhaled toxicants and plays a key role in MCC (Rose and Voynow, 2006). The gel-forming mucins, MUC5AC and MUC5B, are the major macromolecule components of airway mucus. Changes in their production and secretion, as well as in their relative proportion, have been reported in patients with COPD (Hauber et al., 2006; Livraghi-Butrico et al., 2016; Rose and Voynow, 2006). Assessment of changes in these two mucins, therefore, may provide useful information indicative of the toxicity of test articles. In this study, we observed consistent reduction of MUC5AC secretion 24 h after each exposure, whereas secretion of MUC5B was increased by the highest dose but only after the first exposure. However, similar to CBF, the secretion of MUC5AC returned to the baseline level within 72h post-treatment. The intracellular production of MUC5AC and MUC5B was not altered by DHA exposure, suggesting that the decrease in MUC5AC secretion is not the result of the downregulation of mucin expression. The sudden increase in MUC5B secretion may be seen as a compensatory mechanism for the decrease in MUC5AC secretion. Following repeated DHA treatment, it is possible that the cells become adapted and no longer need compensation in mucin secretion from MUC5B. Any adaptation that occurred during DHA treatment did not alter the density and morphology of the mucin-secreting goblet cells significantly. It is known that inflammatory mediators modulate mucin homeostasis (Elkington and Friedland, 2006; Greenlee et al., 2007). To explore the possible mechanism accounting for the temporary disruption of mucin homeostasis, we measured the secretion of 40 human cytokines and chemokines after the first and fifth exposures. However, none of the inflammatory mediators showed significant changes in response to DHA exposure (data not shown). A study by Deshmukh et al. reported the involvement of MMP-9 in mediating MUC5AC expression (Deshmukh et al., 2005). Secretion of MMP proteins (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, and MMP-13), therefore, was quantified. Concentrations of two MMPs, MMP-10 and MMP-13, were found to be temporarily decreased after a single DHA treatment; their downregulation, however, was reversed to baseline level after 5 weekly treatments. MMP-10 is known to play a beneficial role in response to acute infection by modulating the pro-inflammatory responses of resident and infiltrating macrophages (McMahan et al., 2016). Downregulation of MMP-10 increases the susceptibility of mice to airway infections (McMahan et al., 2016). MMP-13 is involved in inflammatory responses and tissue remodeling in several lung diseases (Grzela et al., 2016). The downregulation of both MMP proteins may have temporarily compromised the pro-inflammatory responses of the DHA-treated ALI cultures, as well as caused the disturbance of mucin homeostasis. However, the temporary nature and small magnitude of the responses suggest that DHA exerts marginal effects, if any, on MCC in the ALI cultures. Existing information on the toxicity and mutagenicity of DHA reported in different studies is conflicting (Gallagher, 2018; Goldman and Blaney, 1962). Most studies evaluate its adverse effects on skin cells,
Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the Food and Drug Administration or the National Toxicology Program positions or policies. Funding This study was funded by the National Toxicology Program under an Interagency Agreement between FDA and NIEHS (FDA IAG #22417-0502 and NIH IAG #AES12013). Acknowledgments The authors thank Dr. Paul Howard for bringing up the health concerns of DHA to our attention. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.tiv.2019.04.007. References Barnes, P.J., 2008. Immunology of asthma and chronic obstructive pulmonary disease. Nat. Rev. Immunol. 8, 183–192. BéruBé, K., Aufderheide, M., Breheny, D., R, C., Combes, R., Duffin, R., Forbes, B., Gray, A., I, H., M, K., et al., 2009. In Vitro Models of Inhalation Toxicity and Disease. 37. ATLA Alternatives to Laboratory Animals, pp. 89–141. Biondi, P.A., Passerò, E., Soncin, S., Bernardi, C., Chiesa, L.M., 2007. Selective determination of dihydroxyacetone in self-tanning creams by HPLC as Pentafluorobenzyloxime derivative. Chromatographia 65, 65–68. Cao, X., Lin, H., Muskhelishvili, L., Latendresse, J., Richter, P., Heflich, R.H., 2015. Tight junction disruption by cadmium in an in vitro human airway tissue model. Respir. Res. 16, 30–44. Cao, X., Muskhelishvili, L., Latendresse, J., Richter, P., Heflich, R.H., 2017. Evaluating the toxicity of cigarette whole smoke solutions in an air-liquid-Interface human in vitro airway tissue model. Toxicol. Sci. 156, 14–24. Cao, X., Wang, Y., Xiong, R., Muskhelishvili, L., Davis, K., Richter, P.A., Heflich, R.H., 2018. Cigarette whole smoke solutions disturb mucin homeostasis in a human in vitro airway tissue model. Toxicology 409, 119–128. Chortarea, S., Clift, M.J.D., Vanhecke, D., Endes, C., Wick, P., Petri-Fink, A., RothenRutishauser, B., 2015. Repeated exposure to carbon nanotube-based aerosols does not affect the functional properties of a 3D human epithelial airway model. Nanotoxicology 9, 983–993. Chortarea, S., Barosova, H., Clift, M.J.D., Wick, P., Petri-Fink, A., Rothen-Rutishauser, B., 2017. Human asthmatic bronchial cells are more susceptible to subchronic repeated exposures of aerosolized carbon nanotubes at occupationally relevant doses than healthy cells. ACS Nano 11, 7615–7625. Costa, D.L., 2008. Alternative test methods in inhalation toxicology: challenges and opportunities. Exp. Toxicol. Pathol. 60, 105–109. Deshmukh, H.S., Case, L.M., Wesselkamper, S.C., Borchers, M.T., Martin, L.D., Shertzer, H.G., Nadel, J.A., Leikauf, G.D., 2005. Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation. Am. J. Respir. Crit. Care Med. 171, 305–314. Dvorak, A., Tilley, A.E., Shaykhiev, R., Wang, R., Crystal, R.G., 2011. Do airway epithelium air–liquid cultures represent the in vivo airway epithelium transcriptome? Am. J. Respir. Cell Mol. Biol. 44, 465–473. Elkington, P.T.G., Friedland, J.S., 2006. Matrix metalloproteinases in destructive pulmonary pathology. Thorax 61, 259–266. Gallagher, M., 2018. Exposure to dihydroxyacetone in sunless tanning products: understanding the risks. J. Dermatol. Nurses' Assoc. 10, 11–17. Garone, M., Howard, J., Fabrikant, J., 2015. A review of common tanning methods. J.
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