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Sequential air–liquid exposure of human respiratory cells to chemical and biological pollutants

Sequential air–liquid exposure of human respiratory cells to chemical andbiological pollutants Charles Persoz , Christopher Leleu , Sophie Achard , Magali Fasseu , Jean Menotti ,Pascale Meneceur , Isabelle Momas , Francis Derouin , Nathalie Seta a Université Paris Descartes, Laboratoire de Santé Publique et Environnement – EA 4064, Paris, Franceb Université Paris Diderot, Sorbonne Paris Cité, EA 3520, Paris, Francec Centre de recherche Bichat-Beaujon, U773, Inserm, Paris, Franced AP-HP, Hôpital Saint-Louis, Parasitologie-Mycologie, Paris, Francee AP-HP, Hôpital Bichat, Biochimie, Paris, France Although indoor air has wide ranging effects on human health, the effects of environmental, chemical, and biological pollutants on the respiratory system are not fully understood. In order to clarify the health effects of airborne pollutant exposure, it would appear that toxicological evidence is needed to com- plement epidemiological observations to support by providing biological plausibility. The aim of this study is to manage air–liquid successive exposures to different pollutants such as a chemical pollutant(formaldehyde – FA), and a biological contaminant (Aspergillus fumigatus – Asp) using our in vitro model.
Human alveolar cells (A549) were exposed at the air–liquid interface in an exposure module, firstly to an environmental level of FA (50 ␮g/m3) (or air) for 30 min, and 14 h later to Asp (7 × 108 spores/m3) (or air) for 30 min. After 10 h post-incubation, cellular viability was assessed. Inflammation biomarkers (IL-8, MCP-1) were assayed by ELISA and by RT-PCR.
Whatever the conditions, no cytotoxic effect was observed. FA followed by air exposure did not induce modification of production and expression of cytokines, confirming results with a unique FA exposure. Airfollowed by Asp exposure tended to induce IL-8 expression whereas IL-8 production tended to increaseafter FA and Asp exposure compared to FA and air exposure. The reaction of cells to sequential exposureto FA and Asp was moderate. These results show the feasibility of our model for sequential exposuresto different types of environmental pollutants, allowing using it for preliminary assessment of cellularactivity modification induced by airborne contaminants.
2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
The effects of environmental chemical and biological com- pounds and their association with human respiratory health, Indoor air has important and wide ranging effects on human health and work efficiency, due to the fact that humans are perma- For example, several studies have reported a relation nently inhaling a complex mixture containing numerous pollutants between formaldehyde (FA) exposure at environmental level and asthma, asthma symptoms or airway inflammation (This association is notclearly established, however, with discrepancy in the conclusions(Similarly, as for ∗ Corresponding author at: Laboratoire de Santé Publique et Environnement – EA4064, Faculté de Pharmacie Université Paris Descartes, 4, avenue de are frequently found in indoor environment l’Observatoire, 75006 Paris, France. Tel.: +33 1 53 73 97 28; fax: +33 1 43 25 38 numerous studies have reported an association between mold exposure in the home and asthma symptoms but some disagree on the consequences of exposure to fungal spores. For instance, one current child case–control study has reported asthma cases having significantly higher exposures to 1 Both authors contributed equally to this work.
recently that children living in a farm and exposed 0378-4274/$ – see front matter 2011 Elsevier Ireland Ltd. All rights reserved.
doi: C. Persoz et al. / Toxicology Letters 207 (2011) 53–59 to a wider range of fungi than the reference group, were partly chambers which can hold three separate inserts. The module is equipped with a protected against asthma. This demonstrates the complexity of heated water jacket to keep cells and culture medium at a steady temperature of interaction between these pollutant exposures and induced respi- 37 ◦C. For both FA and Asp experiments, the test atmosphere was delivered on thecellular apical side at a flow rate of 5 ± 0.1 mL/min.
ratory health effects, and it would appear epidemiological studies FA atmosphere was generated as previously described The need to be complemented by toxicological studies in order to chosen dose for these experiments (50 ␮g/m3) has been formerly determined as the fully understand the health effects of airborne pollutant exposure For experiment with Aspergillus spores (Asp), Asp atmosphere (7 × 108 spores/m3) was generated as follows. A genetically modified strain of A. fumigatus Beside experimental toxicological studies traditionally based on expressing a red fluorescent protein, DsRed (CBS 144.89/DsRed) ( complete animal tests, in vitro methods using cell culture technolo- used, kindly provided by J.P. Latgé, Institut Pasteur, Paris, France.
gies are currently being developed and implemented in modern This strain was grown on Sabouraud-Chloramphenicol agar Petri dishes at 37 ◦C for toxicology (Indeed the 3Rs principles that 7 days. Then conidia were harvested by rinsing the culture with sterilized water promote alternatives to animal experimentation are now always containing 0.002% Tween 80. The collected suspension was centrifuged at 3000 × gfor 15 min, then the pellet was resuspended in water containing 0.002% Tween 80 encouraged, due notably to ethical reasons and cost and filtered through a sterile sieve to eliminate remaining agar debris. The purified Classical in vitro exposure methods of airborne contaminants, spore suspension was counted using a Malassez hemacytometer and adjusted to a also called indirect or submerged exposure methods, consist of final concentration of 108 conidia/mL. The viability of collected conidia was checked cell cultures exposed to test pollutants dissolved or suspended in by subculture of serial dilutions of each conidial preparation.
The conidia suspension obtained was aerosolized using a Collison Nebulizer culture medium, like particles collected by impaction or filtration (BGI, USA) provided by compressed air at 2-bars pressure. In preliminary experi- methods, or gas dissolved by bubbling through buffered saline solu- ments, using a particle counter we checked that such conditions allowed generating an aerosol containing >70% of particles ranging between 1 and 5 ␮m, i.e. compris- however do not really reflect human exposure, and are not there- ing the 2.5–3 ␮m size of Aspergillus spores. Less than 5% of particles >5 ␮m were fore suitable for gaseous pollutant testing.
detected suggesting that few spores clustered during nebulization. The generatedaerosol was connected to the Vitrocell® exposure device, resulting in exposure of Methods which better mimic the inhalation process and contact the cellular apical side of cell culture.
between epithelial cells and air are considered to be more relevant In order to check the conidial concentration at the cell culture level, a sample than the in vitro exposure methods above. Two main direct expo- of air was collected in the Vitrocell® inlet using a vacuum pump at a flow rate of sure approaches are available: the first is intermittent exposure 10 L/min during 1 min then passed through a 0.8 micron size filter (AAWP, Millipore).
The filter was then incubated into 1 mL of saponin 2% during 15 min and vortexed to (cells are exposed to gaseous compounds and culture medium at elute the collected conidia. These were enumerated in a Malassez hemacytometer.
regular intervals using rocker platforms or rolling bottles), and the Three repeated measurements were performed for each exposure experiment.
second is continuous exposure. In this latter case, cells culturedon an insert made of microporous membranes are continuously 2.3. Cell line, culture conditions and cell exposures exposed to airborne pollutants on their apical side for a period oftime, whilst their basolateral side is in contact with the culture Human alveolar epithelial cells, A549 (CCL-185 purchased from ATCC, VA, USA) were cultured as previously described (Cells (at the density of20,000 cells per insert) were grown on inserts (1.13 cm2). Passages of cells were certain tools have recently been developed to perform air–liquid between 3 and 15 for all experiments. Culture medium (DMEM:F12 supplemented exposure to environmental pollutants, most have been used for sin- with 10% FCS) was added on both sides: 300 ␮L on the apical side, and 1000 ␮L on gle period exposure to an unique pollutant at levels far above the the basolateral one, and incubated 72 h before experimentation at 37 ◦C and 5% CO2.
Just before exposure, culture medium was removed and replaced with DMEM:F12 medium supplemented with 1% FCS. Cells were then exposed at the air–liquidinterface for 30 min to either air or pollutants. the experimen- tal protocols. Protocol A includes unique exposure to air or FA followed by 14 h such an in vitro model for formaldehyde single period exposure of post-incubation. Protocol B consisted of two sequential exposures of 30 min each lung epithelial cells (However, in these exper- (exposures 1 and 2): the first to air or FA and the second to air or Asp. In this case, a imental conditions, the pollutants studied were always chemical 10 h post-incubation followed the second exposure. Four experimental conditionswere thus obtained as shown in B).
rather than microbiological. If we are to mimic real atmospheric Prior to each exposure the culture medium at the apical side was removed to exposure then cells must be exposed for longer, or more frequently, allow a direct contact between the cells and the tested pollutants. After each expo- sure, 300 ␮L of culture medium with 10% FCS were added at the apical side and In this context, the aim of this study is to manage air–liquid cells were post-incubated for 14 h (exposure 1) and 10 h (exposure 2) at 37 ◦C, 5% successive exposures to different pollutants such as a chemi- CO2. At the end of each post-incubation, culture medium was removed and storedat −20 ◦C for cytokine assay and at 4 ◦C for LDH assay. The cell monolayer was used cal pollutant (formaldehyde – FA), and a biological contaminant for RT-qPCR at the end of the experiment.
(Aspergillus fumigatus – Asp) using our in vitro model. These, both Control cells introduced at each experiment corresponded to cells cultured in known to be frequently simultaneously detected in domestic envi- submerged conditions without any exposure. Each experiment was realized four times in triplicates with an interval between two tests of one week.
2. Methods
Membrane integrity was determined by measuring the release of intracellu- lar lactate dehydrogenase (LDH) into the supernatant medium. LDH was measured using an in vitro assay kit in which LDH oxidizes the substrate l-lactate buffered at penicillin–streptomycin and trypsin were purchased from Gibco via Invitro- a pH of 9.4 in the presence of NAD+ to yield pyruvate and NADH,H+ which absorbs gen (WI, USA). Formaldehyde, TNF␣, Tween 80 and saponin were obtained from light at 340 nm. Concentrations were initially expressed as unity of consumed sub- Sigma–Aldrich (MO, USA). The LDH assay kit (K2054) was from Siemens and used strate per liter (U/L) and cellular viability, expressed as a %, was calculated with with an automaton Dimension Vista 1500 (Siemens Healthcare Laboratories). The sample concentration and maximal LDH activity (positive control obtained by cell DuoSet kits for ELISA assays (DY208 and DY271) were produced by R&D Systems lysis after 2 h Triton X-100 1% treatment), each normalized on control cells concen- (MN, USA). Inserts and flasks for cell culture were provided from Greiner Bio-One tration Control cell LDH concentration was 25 ± 4 U/L (n = 4) and maximal activity was 1652 ± 237 U/L (n = 4).
2.2. Atmosphere generation and cell exposure device Local cellular inflammation was assessed by quantification of Interleukin-8 The Vitrocell® device used for exposure experiments has been previously (IL-8) and Monocyte Chemoattractant Protein 1 (MCP-1) concentrations in the described by this device consists of three exposure apical culture supernatants of A549 cells using ELISA assay kits following the C. Persoz et al. / Toxicology Letters 207 (2011) 53–59 Cell viabilityCytokines producƟon and expression Exposure 2
Nomenclature for condiƟons in protocol B
Exposure 1
Fig. 1. Exposure protocol.
manufacturer’s instructions as stated previously. Results were expressed as a ratio were confirmed using nonparametric Mann–Whitney U test. Difference was signif- of cytokine production (± standard error) compared to the one of the control cells.
3. Results
mRNA extraction and purification were performed with Qiagen RNeasy® plus mini kit (Qiagen, Germany) according to the manufacturer’s protocol for culturedcells. Samples were examined for concentration with the NanoDrop® ND-1000 Spec- trophotometer (NanoDrop Technologies, USA) and for integrity with the RNA 6000Nano LabChip® kit and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). All To apply our in vitro model to sequential exposure to a chemical the RNA Integrity Numbers of the samples were >7. RNA was then stored at −80 ◦C pollutant and molds, two limiting factors concerning post-exposure cDNA synthesis was performed using the cDNA verso® kit from Thermo Scien- incubation time had to be taken into account: limiting the cell line tific according to the manufacturer’s specifications. Reverse transcription products growth to 24 h, and avoiding the germination of conidia.
were amplified using a Thermal Cycler 2720 (Applied Biosystems). Primers for In a preliminary work, conidia germination was monitored at cytokines (IL-8: Hs00174103 m1, MCP-1: Hs00234140 m1) and reference genes different times after Asp exposure, and showed that an incubation (GAPDH: Hs99999905 m1, B2M: Hs00984230 m1, HPRT1: Hs01003267 m1) were time of 10 h resulted in germination and limited hyphal growth obtained from Applied Biosystems. The real-time PCR run on the LightCycler® 480Real-Time PCR System (Roche Applied Sciences, Basel, Switzerland). Each gene was whereas a longer incubation time resulted in a deleterious fun- tested in duplicate within the same PCR run on an additional 96-well plate. After gal invasion of the culture (Supplementary data, We being diluted by ten, cDNA (5 ␮L) from each reaction for all genes was added to a therefore considered 10 h as the maximal incubation time after Asp PCR reaction mix containing 1X TaqMan® Universal Master Mix II without Uracil-N- spore exposure and scheduled this exposure after FA exposure.
Glycosylase and 1 ␮L Endogenous Control assay or Gene expression Assay in a 20 ␮Lreaction volume. Standard cycling conditions were used [95 ◦C for 10 min, (95 ◦C for For the repeated exposure protocol, given cell confluence was 15 s, 60 ◦C for 60 s) ×40 cycles].
reached after 24 h and maximum 10 h post-incubation after Asp The expression levels obtained were normalized against the reference gene, exposure, post-incubation after chemical pollutant exposure, being and changes in expression levels were given relative to the untreated controls.
the first exposure, had to be 14 h. We compared the results after Data quantification normalization of expression of marker genes was achieved by the first exposure with our previous results, where experimen- normalization against a subset of three reference genes on the array that did notshow significant changes in expression under our experimental conditions (B2M tal conditions were designed with 24 h of post-incubation after (␤2-microglobulin), HPRT (hypoxanthine phosphoribosyltransferase) and GAPDH air–liquid exposure After 14 h post-incubation (glyceraldehyde 3-phosphate dehydrogenase)).
air-exposure induced an increase of 42.6 ± 6.9% of IL-8 production Stability of the 3 reference genes was assessed with the geNorm applet compared to the control cells (p < 0.01), and MCP-1 production was (). The Cq values were transformed toquantities using gene specific efficiencies, according to the geNorm manual. Gene not modified, similar to exposure followed by 24 h post-incubation expression stability (M) was calculated and the genes were ranked from best to (In the same way, FA did not significantly mod- worst, based on the M value. The gene with the highest M, i.e. the least stable ify IL-8 (ratio to control cells = 1.2 ± 0.3 vs. 1.4 ± 0.1 for air) and gene, was then excluded in a stepwise fashion until the most stable genes were MCP-1 (ratio = 0.6 ± 0.1 vs. 0.8 ± 0.1 for air) production by A549 cells after 14 h incubation. Although experimental post-incubation time Cq, the quantification cycle number at which the amount of amplified target reaches a fixed threshold was determined (default threshold was reduced, the cellular response was similar to that previously settings were used in all instances). The cycle number above which the Cq was considered as a false positive (cycle cutoff point) was set up at 35. 2− Results were expressed as arithmetic mean of 2− three independent experiments after normalization on the most stable gene in ourexperiments, HPRT.
3.2.1. Sequential exposure to air (Air–Air) To validate sequential exposure, we first studied the cellu- lar effects of repeated exposure to the same gas, air. The doubleexposure to air (protocol B: Air–Air) did not impact cell Statistical analyses were performed using R software version 2.12.2 project.org). The difference between two conditions was considered using Student’s viability and only slightly modified the inflammation parame- t-test for cellular viability, cytokines production and cytokines expression. Results ters. Indeed that cell viability (2A) and IL-8 hourly C. Persoz et al. / Toxicology Letters 207 (2011) 53–59 Fig. 2. Cell viability (A) and IL-8 production per hour (B) of A549 cells after a single or repeated exposure to air (n = 4).
production (2B) of A549 cells after one or two exposures to air on the inflammatory response caused by FA. This sequential expo- were not modified by the number of exposures to air. Similarly sure to FA and Asp (FA–Asp) did not alter cell viability (96.5 ± 3.2%) IL-8 expression, evaluated by RT-qPCR, was not modified between when compared to FA–Air exposure (96.6 ± 2.0%).
Cq = 2.41 ± 0.92 vs. 2.47 ± 0.76, respec- The reaction of cells to sequential exposure to FA and Asp was tively). Considering MCP-1, the production of this cytokine was not moderate. Indeed FA–Asp induced an increase, although this did not modified by sequential air-exposure, but its expression was sig- reach significance, of IL-8 production (p = 0.10) when we compared Cq = 1.05 ± 0.13 vs. 1.65 ± 0.17, p < 0.05, these results with those obtained with FA–Air A slight respectively after one and two exposures).
trend, but not significant, was also observed for the expression These results indicate that cells reacted to one or two air expo- of this cytokine Finally, production of MCP-1 tended to decrease whilst its expression tended to increase (p = 0.16) (and D).
3.2.2. Sequential exposure to air and Aspergillus fumigatus(Air–Asp, grey) 4. Discussion
In order to represent a complex environmental exposure, we also exposed cells to a biological contaminant, spores of fungal In industrialized countries, about 90% of our time is spent species, Asp. Because of the limiting factors related to the use of indoors where there are numerous pollutants, including chemical such a pollutant and the duration of total incubation, cells were and biological contaminants, to which we are chronically exposed exposed to Asp as the second exposure pollutant, after an initial The difficulties in representing environmental exposure to air. In our experimental conditions, cell viability at exposure in vitro are: (1) establishing the duration and repetition the end of experiment was not altered (96.2 ± 3.7% vs. 92.4 ± 1.9% of exposure, (2) assessing the variety of real exposure, associat- for Air–Air). IL-8 production did not change (and a slight ing chemical, biological and physical compounds but not significant trend was observed for IL-8 expression (p = 0.21) and (3) selecting the chosen levels for tested pollutants.
compared to Air–Air exposure. As regards MCP-1 the pro- In the present study, FA was chosen to typify chemical pol- duction in supernatant was unchanged in comparison to Air–Air lution: it is a major indoor air pollutant and targets mainly the exposure and MCP-1 expression was not modified after of FA used in our experiments (50 ␮g/m3) was chosen according tolevels typically observed in the domestic environment ( 3.2.3. Sequential exposure to FA and Air (FA–Air, grey) A549 cells were next exposed, first to FA at 50 ␮g/m3 and tamination, on the other hand, was represented using the species then to Air. At the end of experiment, no impact on viability Aspergillus fumigatus. This fungi is frequently found in the indoor was observed when we compared Air–Air and FA–Air (92.4 ± 1.9% and 96.6 ± 2.0%, respectively). Results concerning the inflamma- known for its numerous respiratory health effects ( tory response showed a decrease of production of IL-8 (and The concentration chosen for our study is typical of that found at the MCP-1 (but both were non-significant (p = 0.17; p = 0.30).
high end of concentrations in contaminated indoor environments The expression of these two cytokines was not modified The use of these pollutants has been previously reported, Taking these results together, that is sequential exposure to air although more often in submerged conditions ( with or without another pollutant (Air–Air, Air–Asp and FA–Air), we confirmed that a second exposure did not induce deleterious or pared to airborne levels. Some authors have reported FA or Asp specific effects on A549 cell behavior.
atmosphere generation for in vivo studies, but levels were oftenhigh (Some others 3.2.4. Sequential exposure to FA and Asp: (FA–Asp, Finally, we tested the combination of exposures to FA and Asp, in order to study the synergic effects of a microbiological contaminant and 250 ␮g/m3 respectively) that were relevant compared to C. Persoz et al. / Toxicology Letters 207 (2011) 53–59 Fig. 3. Production (A) and expression (B) of IL-8 and MCP-1 (C and D) by A549 cells for each condition of the exposure experimental protocol. Productions are presented as
a ratio of unexposed cells and expressions are normalized on HPRT gene (n = 4).
occupational exposures, but far above the concentrations found in post-incubation, as previously described (In domestic environment (mainly <100 ␮g/m3) addition, we refined the study of inflammatory response to expo- In this context, we paid very careful attention to the gener- sures by assessing the expression of these cytokines at the end of ation of realistic pollutant levels and to the checking of these levels the exposure. Testing release as well as de novo synthesis helped to describe the early inflammatory response of the cells more com- Since environmental exposure is rarely a one-off event, we repeatedly exposed cells to FA and Asp in our cellular model. To the The first issue in our experimental protocol was to make sure best of our knowledge, this is the first time that such an air–liquid that the shortening of the first post-incubation time, from 24 h sequential exposure has been performed. In addition, we made to 14 h, did not profoundly modify cellular response to gas expo- sure that our protocol overcame certain problems inherent in the sure. For viability as well as cytokine production, we obtained an design. We wanted to sequentially expose A549 cells to the two analogous response, and once again observed the increase of IL-8 pollutants during a 24 h cycle after the first exposure, in order to production after air exposure and equivalent levels between air and maintain suitable conditions as previously defined (seeding and cellular monolayer growth) (The choice of expo- The second issue concerned the feasibility of repetitions, since sure sequence and duration of exposure to Asp was determined cells exposed at the air–liquid interface undergo a certain degree by kinetics of germination and hyphal development ( of stress due to the air flow of exposure itself, this during even a single exposure (Previous research into cell tact between epithelial cells and spores until the early stages of viability has shown that one exposure of 60 min induced cellular germination and hyphal growth. Maintaining cell culture over 10 h damages (contrarily to two exposures of 30 min after spore exposure would however lead to uncontrolled fungal with an in-between period of 14 h. Considering cytokines, a second growth that could induce cellular damage and markedly bias the exposure to air did not induce a supplementary increase of IL-8 pro- interpretation of the observed results. Considering this constraint duction or expression whereas MCP-1 production and expression, FA exposure was performed before Asp.
not modified after the first air exposure, were slightly increased Our purpose was to set up an in vitro model to assess the impact of repeated exposures to airborne pollutants at environ- Results from the other exposure combination data led to the mental levels on cell inflammation, and we therefore had to avoid same conclusion: no cytotoxicity and no major stress. Indeed, when any cytotoxic condition: whatever the conditions, no cytotoxic repeated exposures were performed with other compounds in effect was observed. The biomarkers selected to study the inflam- addition to air, either FA or Asp, none induced cell toxicity. Although mation response were two chemokines that play a major role in cellular response was always modest and indeed should be con- the inflammatory process of the respiratory tract, IL-8 and MCP-1, firmed by further experiments, repeated exposure was feasible in respectively neutrophil and monocyte chemoattractants our experimental conditions, with cells keeping their metabolic We assessed their production in apical supernatants during C. Persoz et al. / Toxicology Letters 207 (2011) 53–59 The third and last issue dealt with the feasibility of cell exposure Appendix A. Supplementary data
to fungal spores. To the best of our knowledge this is the first timethat fungal contaminants have been used in air–liquid exposure Supplementary data associated with this article can be found, in experiments. Fungal conidia are usually in contact with respiratory cells by suspension Interestingly, Asp spore exposure coupled with prior air exposurewas not cytotoxic, even after 10 h post-incubation and early hyphal References
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