Polycyclic aromatic hydrocarbon components contribute to the mitochondria-antiapoptotic effect of fine particulate matter on human bronchial epithelial cells via the aryl hydrocarbon receptor
© Ferecatu et al. 2010
Received: 30 April 2010
Accepted: 21 July 2010
Published: 21 July 2010
Nowadays, effects of fine particulate matter (PM2.5) are well-documented and related to oxidative stress and pro-inflammatory response. Nevertheless, epidemiological studies show that PM2.5 exposure is correlated with an increase of pulmonary cancers and the remodeling of the airway epithelium involving the regulation of cell death processes. Here, we investigated the components of Parisian PM2.5 involved in either the induction or the inhibition of cell death quantified by different parameters of apoptosis and delineated the mechanism underlying this effect.
In this study, we showed that low levels of Parisian PM2.5 are not cytotoxic for three different cell lines and primary cultures of human bronchial epithelial cells. Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin) by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes (cell size decrease, massive chromatin and nuclear condensation, formation of apoptotic bodies). The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion. Experiments performed with different compounds of PM2.5 suggest that endotoxins as well as carbon black do not participate to the antiapoptotic effect of PM2.5. Instead, the water-soluble fraction, washed particles and organic compounds such as polycyclic aromatic hydrocarbons (PAH) could mimic this antiapoptotic activity. Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR) showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM2.5 at the mitochondrial checkpoint of apoptosis.
The PM2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state induced after pollution exposure and might delay repair processes of injured tissues.
calcium ionophore (calcimycin)
aryl hydrocarbon receptor
chronic obstructive pulmonary diseases
Diesel exhaust particles
3, 3 dihexyloxacarbocyanine iodide
epidermal growth factor
granulocyte monocyte colony-stimulating factor
- H2O2 :
mitochondrial membrane permeabilization
polycyclic aromatic hydrocarbons
reactive oxygen species
small interfering RNA
mitochondrial transmembrane potential.
Nowadays, air pollution is considered as a major inducer of harmful health effects, especially due to fine particulate matter (PM2.5, atmospheric particles with an aerodynamic diameter equal or less than 2.5 μm). Urban PM2.5 is a mixture composed mainly of soots from fossil fuel combustion  together with several components adsorbed, including organic elements, biological species and metals . In vitro short-term exposure to PM is associated with an inflammatory response as a consequence of cellular oxidative stress increase . Fine PM are taken up by airway epithelial cells and alveolar macrophages [4, 5] leading to proinflammatory cytokine expression and release (i.e. GM-CSF, IL-1α, IL-8, TNFα, etc) [6, 7] as well as the production of reactive oxygen species (ROS) . Moreover, recent data demonstrate that short exposure of bronchial or nasal epithelial cells to urban PM2.5 provokes the secretion of EGFR ligands and Amphiregulin, which leads to GM-CSF secretion via an autocrine pathway .
Long-term effect of atmospheric particles remains underestimated. Nevertheless, epidemiological studies provide evidence of their deleterious impacts by increasing cardiopulmonary morbidity and mortality , asthma , bronchitis , exacerbation of chronic obstructive pulmonary disease (COPD, ). In addition, cancerous pathologies such as tracheal, bronchial and lung tumors are exacerbated . In tissues, chronic exposure was associated with persistence of particles into the lungs leading to bronchioli wall thickening  and airway remodeling characterized by epithelial mucus-producing cells metaplasia, subepithelial fibrosis and airway smooth muscle hypertrophy/hyperplasia as observed in chronic asthma and COPD . Thus, mechanisms involved in airway remodelling might be the excessive cell proliferation as well as the resistance to the apoptotic cell death.
Apoptosis is a programmed cell death defined by specific morphological alterations but with only slight ultrastructure modifications of cytoplasmic organelles and phosphatidylserine (PS) residue externalization . It is noteworthy that mitochondrial alterations constitute the checkpoint of the apoptotic cell death. This is highlighted by the mitochondrial membrane permeabilization (MMP) which is measured by the decrease of mitochondrial transmembrane potential (ΔΨm), and by the subsequent superoxide anion production and Cytochrome c release. The activation of caspases or other proteases triggers the proteolysis of specific substrates involved into the final appearance of morphological features of apoptosis. Most publications dealing with toxicity of airborne particles showed an induction of apoptosis associated with ROS generation, ΔΨm drop, caspase-9 activation and DNA fragmentation . In vitro experiments showed that PM-induced apoptosis was reported in normal human lung tissue or airway epithelial cells [19, 20].
The toxicity of ambient particles is mainly attributed to various adsorbed components. For instance, organic compounds are known to mimic the apoptotic effect of PM in various cell types through pathways which require the activation of the aryl hydrocarbon receptor (AhR) and the generation of ROS leading to DNA damage. Nevertheless, polycyclic aromatic hydrocarbon (PAH) induced-apoptosis is mainly mediated via the mitochondria pathway in a p53-dependent manner . Metals also affect human health, especially when these toxicants compete with essential elements and modify many cellular processes. Transition metals promote apoptosis through ROS generation, mitochondria dysfunction, activation of MAPK, p53 and caspases or down regulation of antiapoptotic proteins Bcl-2 . Metals and the water-soluble fractions of PM are known to cause inflammation and cancer mostly due to DNA damage as a consequence of ROS generation by Fenton reaction. In addition, the exacerbation of asthma after inhalation of PM is mainly attributed to the biological compounds. Endotoxins induce proinflammatory cytokines production  and are able to provoke apoptosis-like cell death involving a scavenger receptor.
Most of PM pro-apoptotic data were obtained in vitro from acute exposure (with 80 to 100 μg/cm2 of particles) which usually corresponds to high pollution periods. The purpose of the present study was to investigate the effect of low doses of air particles (PM2.5), on different bronchial epithelial cells (tumoral, immortalized and primary cells) regarding their induction or reduction of apoptosis. First, we found that Parisian PM2.5 are not cytotoxic, but have an antiapoptotic effect towards well-known cell death inducers, A23187, staurosporine and oligomycin. The reduced apoptosis observed after particle exposure is not related to the pro-inflammatory response and the EGF pathway. Moreover, water-soluble as well as organic components such as heavy PAH, are able to mimic the effects triggered by PM2.5, suggesting that such compounds are involved in the antiapoptotic effect. Finally, we identified the aryl hydrocarbon receptor as a molecular effector involved in the mechanism of the antiapoptotic effect of PM2.5 on human bronchial epithelial cells.
PM2.5 are not cyctotoxic in human bronchial epithelial cells
These results might be related to the batch of PM2.5 used, in particular timing and location of particle collection. To test this hypothesis, we used several batches of Parisian PM2.5: Auteuil-Winter (AW), Auteuil-Summer (AS), Vitry-Winter (VW) or Vitry-Summer (VS) collected in the Paris area: (i) Porte d'Auteuil adjacent to a major highway and considered as a curbside station and (ii) a school playground at Vitry-sur-Seine in the suburb of Paris. When bronchial cells were exposed 24 h to these PM2.5 (10 μg/cm2), we noticed only an increased granularity corresponding to particle uptake without any reduction in cell size (data not shown). Apoptotic cell death was then quantified by ΔΨm loss and plasma membrane permeabilization, and none of these parameters was significantly increased by exposure to the four different batches of PM2.5 (Figure 1D). Altogether, Parisian PM2.5 seem to have no cytotoxic effect in several human bronchial epithelial cells, including the primary NHBE cells.
Parisian PM2.5 have an antiapoptotic effect
The antiapoptotic effect of PM2.5 is related to organic and water-soluble components
The antiapoptotic mechanism is mediated by the aryl hydrocarbon receptor
Finally, we tested the effect of AhR silencing in the antiapoptotic effect observed after PM2.5-exposure. For this, we used validated fluorescent-siRNA (Alexa Fluor 647) in order to select the fluorescent positive cells by flow cytometry. After siRNA optimization (80% transfected cells with 10 nM siRNA after 48 h, data not shown) and validation of AhR silencing by western blot (Figure 7B upper panel), DiOC(6)3 and PI assays were performed by flow cytometry on cells exposed or not to PM2.5 and/or A23187 for 24 h as before. Figure 7B (lower graph) shows that AhR silencing significantly reduced the protection triggered by PM2.5 (18% vs. 27% reduction of DiOC(6)3 low) alike the antagonist (alpha-NF) did. Interestingly, both the AhR silencing and AhR antagonist partially reduced the PM2.5-protective effect with almost the same extent (10%). The increase in alpha-NF concentration (20 μg/ml, data not shown) or siRNA-AhR amount (25 nM, Additional file 1 Figure S3) did not completely abolish the protection suggesting that another pathway might be involved. Taken together, these results suggest that AhR partially contributes to the antiapoptotic effect of PM2.5 exposure.
To our knowledge, this article is the first study presenting evidence that low concentrations of PM which are not cytotoxic, have an antiapoptotic effect on human bronchial epithelial cells. We report here the cellular effects of PM2.5 from two sites in Paris, sampled in winter and in summer. In order to remove the risk of cell type-specific events, our study was done in parallel on different human bronchial cell lines as well as on primary cells. We show that the four batches of PM2.5 are not cytotoxic on human bronchial cells, at a range of concentration from 1 to 50 μg/cm2. This is supported by data from flow cytometry, with the measurement of the main apoptotic hallmarks, as well as from electron microscopy data. Our results were obtained with a low concentration of PM2.5 unlike previous publications performed with higher doses (e.g. 100 μg/cm2, ). Indeed, the standard dose used here (10 μg/cm2) is a concentration which could mimic a five day exposure of PM2.5 in the tracheobronchial region, considering that PM2.5 mass deposition is 2.3 μg/cm2/24 h . Our results are in agreement with a previous publication where BEAS-2B human bronchial cells were not susceptible to diesel exhaust particles-induced apoptosis  and here, we provided supplementary evidences of a non-toxicological activity of PM2.5 in NHBE primary culture. Moreover, in our studies and those of Sanchez-Perez et al. , the lack of induced-apoptosis triggered by PM at 10 μg/cm2 suggests that a "sub-lethal" concentration could have different impacts on cell fate than at high concentrations.
The originality of this work is that PM2.5 exposure confers a specific decrease in apoptosis induced by A23187, staurosporine and oligomycin as demonstrated in immortalized (16HBE), cancerous (NCI-H292, BEAS-2B) as well as primary normal bronchial epithelial cells (NHBE). In order to characterize the molecular mechanism of the antiapoptotic activity of PM2.5 exposure, first we demonstrated that the reduction of apoptosis is observed prior to proinflammatory cytokines secretion which led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion by bronchial epithelial cells. However, PM2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state in vivo induced after pollution exposure and might delay repair processes of injured tissues .
To further delineate the mechanism of the antiapoptotic activity, a strategy would be to identify the cellular targets which are in common between staurosporine, A23187 and oligomycin. On one hand, staurosporine and A23187 are known to regulate cellular calcium signaling pathways inducing an endoplasmic reticulum stress which leads to cytoplasmic calcium uptake , mitochondrial Ca2+ overload  and finally ΔΨm drop. Thus, PM2.5 exposure might counteract the Ca2+ uptake induced by these apoptotic inducers. However, this hypothesis is in discrepancy with the fact that the antiapoptotic effects of PM2.5 were not observed when using ionomycin, which is a well-known calcium ionophore, like A23187. Indeed, A23187 and ionomycin, which are both monocarboxylic ionophores, promote a selective increase of cytosolic Ca2+ . But on the contrary to A23187 , a recent study showed that ionomycin did not allow the mitochondrial calcium overload in epimastigote cells of Trypanosoma cruzi . The measurement of cytosolic and mitochondrial calcium uptakes in response to A23187 and ionomycin might allow us to understand why A23187-induced apoptosis is sensitive to PM while ionomycin is not. Moreover, caspases are the main effectors of apoptosis, but A23187, staurosporine and ionomycin can also activate Ca2+-specific proteases, such as calpains [33, 34]. Indeed, our preliminary studies showed that calpains are activated after A23187 treatment of 16HBE and NCI-H292 cells (data not shown). As described for oligomycin, A23187, but not ionomycin, is a specific inhibitor of mitochondrial ATP synthase also known to catalyze the direct exchange of Ca2+/2H+ in liver mitochondria  and to disrupt the mitochondrial transmembrane potential . All these data suggest that ionomycin and A23187 might trigger the apoptotic process by slightly different mechanisms especially at the mitochondrial level. Thus, we hypothesize that PM2.5 could directly reduce apoptosis at the mitochondrial step by maintaining ΔΨm, or via the upregulation of antiapoptotic proteins such as Bcl-2 known to protect from A23187-induced apoptosis [24, 37].
Humans are exposed to a mixture of compounds including organic and inorganic components adsorbed on PM. Evidences suggest that organic compounds such as the polycyclic aromatic hydrocarbons (PAH) can mimic the pro-oxidant  and apoptotic effect of PM . Here, we investigated the role of different organic compounds (PAH, Oex), particles devoid of hydrosoluble components, (wash, CB) and aqueous extracts (Aex) of PM2.5 with respect to cell death. We found that the organic extracts and several heavy PAH, B(a)P in particular, could reproduce the antiapoptotic activity. Moreover, the water-soluble fraction also contributes to the reduction of apoptosis while carbon black, light PAH and endotoxins have no effect. In our study, B(a)P is the compound that protects the most efficiently from apoptosis induced by A23187. This points out a possible link between PM2.5-exposure and the antiapoptotic effect observed herein, as also suggested by Hung et al. . The harmful health impacts of PAH are well-known, like the promotion of cancers. B(a)P-diones, which are photomodified by the sunlight, were also found in air particulate matter. In agreement with our results, a recent work demonstrated that sunlight-exposed B(a)P inhibits apoptosis induced by cell detachment . B(a)P is metabolized by cells, transformed into a reactive intermediate (anti-7,8-dihydrodiol-9,10-epoxy-benzo[a]pyrene, BPDE) that causes DNA damage and mutations in tumor suppressor genes, such as p53 . This toxic metabolite BPDE is also capable to suppress apoptosis of mammary epithelial cells .
The main cellular target of PAH adsorbed on PM is the aryl hydrocarbon receptor (AhR), thus we addressed the question of AhR involvement in the antiapoptotic effect after PM2.5 exposure. We showed here that the activation of AhR by the agonist beta-naphtoflavone improves the antiapoptotic effect. On the contrary, the inhibition of AhR (using a specific inhibitor or RNA silencing) diminished the antiapoptotic effect suggesting that AhR is involved in this process. An additional argument is brought by the absence of antiapoptotic activity when we tested light PAH, which were previously shown to poorly promote AhR activation . AhR is a cytoplasmic ligand-dependent transcription factor which translocates to the nucleus in order to bind specific Xenobiotic Responsive Elements in the promoter of its target genes, leading to the activation of phase I and II metabolizing enzymes and thus contributing to detoxification . But in the absence of ligand, many data suggest other roles than detoxification  and recent evidences suggest that AhR inactivation could modify the expression of numerous genes, including those involved in cell cycle regulation . In accordance with our results, other publications suggest an antiapoptotic activity of AhR by a direct interaction with E2F1 leading to the reduction of E2F1-mediated pro-apoptotic genes expression . This is consistent with the idea that the AhR might modulate cell death at the mitochondrial checkpoint, for instance by upregulating the expression of antiapoptotic bcl-2, bcl-xL, mcl-1 or agr2 genes [48, 49] or by repressing the pro-apoptotic apaf-1 . Moreover, AhR might indirectly regulate apoptosis through the MMP step by increasing the expression of the anti-apototic protein VDAC2  which is known to participate to the permeability transition pore (PTP) and which also bind to and inhibit the apoptotic protein Bak . In the light of our observations, it will be interesting to find out the genes encoding mitochondrial regulators which are modulated by AhR and involved in the protection observed after PM2.5-exposure or B(a)P treatment. It is also important to point out that both A23187 and STS could induce apoptosis via a Ca2+-dependent pathway through mitochondrial PTP opening and that VDAC plays a crucial role in the transport of Ca2+ into this organelle .
Materials and methods
Urban atmospheric PM2.5 were collected during winter or summer 2003 at two locations in Paris: an urban background station at Vitry-sur-Seine (VW: Vitry winter and VS: Vitry summer), a suburb of Paris; and a curbside station at Porte d'Auteuil bordering the highway ring road of Paris (AW: Auteuil winter and AS: Auteuil summer). Particles were recovered on 150 mm diameter nitrocellulose filters (HAWP, Millipore, Saint-Quentin-en-Yvelines, France) with a high volume sampler machine (DA-80 Digitel, Switzerland, flowrate: 30 m3/h, ). Their PAH and metal content have been previously described . PM2.5-AW organic extracts (Oex) were obtained after extraction by dichloromethane, then dried and redissolved in dimethyl sulfoxide (DMSO, Sigma). Oex were used at the concentration found on particles according to the soluble organic fraction (SOF) determined for PM2.5-AW particle sample (10%). The aqueous extract of PM2.5-AW (Aex) containing hydrosoluble components was obtained after the washing of the particle suspension and two centrifugations at 10,000 g, followed by filtration of the supernatant through a 0.22 μm Durapore® filter (polyvinylidine difluoride) (Millex® GV, Carrigtohill, Cork, Ireland). Cells were exposed to a volume of aqueous extract equivalent to the volume of particle suspension used. Particles collected after the two centrifugations constitute the washed PM2.5-AW (wash) devoid of hydrosoluble components. Carbon black particles (CB, Fr101, 95 nm, 20 m2/g) were purchased from Degussa (Frankfurt, Germany). All particles were stored in DMEM medium and used at standard dose 10 μg/cm2 (40 μg/ml). For treatment, after thawing, particles were sonicated three times for 20 s at 70W (Vibracell, Bioblock Scientific, Illkrich, France) and added directly onto the cells. Purified PAH, B(a)P, DB(a,h)A, B(g,h,i)P, iP, B(b)F, PA, FA and vehicle cyclohexane were purchased from Sigma.
Cell culture conditions
Human bronchial epithelial cells 16HBE14o- kindly provided by Dr. D.C. Gruenert (Colchester, VT, USA , were cultured in DMEM/F12 medium (Invitrogen) supplemented with 2 mM GlutaMAX™-I, 100 U/ml penicillin, 100 μg/ml streptomycin, 1.25 μg/ml fungizone and 2% UltroserG (UG, BioSepra, France). Cells were grown to subconfluence on bovine collagen (Purecol Natucan, 0.5 mg/cm2) and human fibronectin coating (Sigma, 4 μg/cm2). Prior to particle treatment, UG was removed. BEAS-2B human bronchial epithelial cells (provided by Dr. J. Boczkowski, Faculté de Médecine Xavier Bichat, Université Paris 7, France) were cultured in LHC-9 medium containing retinoic acid (33 nM). The human lung mucoepidermoid carcinoma cells (NCI-H292) were purchased from the American Type Culture Collection (Rockville, MD) and cultured in RPMI-1640 medium (Invitrogen) supplemented with 1% GlutaMAX™-I and 10% fetal calf serum. Primary cultures of normal human bronchial epithelial (NHBE) cells were obtained from Lonza and cultured for in Clonetics® BEGM medium (Cambrex, Walkersville, MD, USA) supplemented with EGF 25 ng/ml. During treatment NHBE cells were grown in DMEM/F12 without growth factors.
Chemicals and apoptosis measurement
Cells were exposed 4 h to PM2.5 (1-50 μg/cm2) prior to addition of apoptotic inducers for additional 20 hours: rotenone (Rot, 5 μM), antimycin (AMA, 25 μg/ml), oligomycine (Omy, 5 μM), ionomycin (Iono, 0.5 μM), A23187 (3 μM), staurosporine (STS, 1 μM) and hydrogen peroxide (H2O2, 500 μM). All drugs were purchased from Sigma. Apoptotic parameters were quantified by flow cytometry performed on CyAn ADP LX (Dako Cytomentation funded by the Ligue Nationale contre le Cancer R03/75-79) using 3, 3 dihexyloxacarbocyanine iodide (DiOC6(3), 2 nM) for ΔΨm quantification, 10 μg/ml propidium iodide (PI) for determination of plasma membrane permeabilization, 2 μM hydroethidine (HE, Molecular Probes) for superoxide anion generation, and Annexin V conjugated with fluorescein isothiocyanate (FITC, Sigma) for the assessment of phosphatidylserine (PS) exposure . Percentage of induction of apoptosis is calculated according to the following formula: % = 100 × (% of apoptotic treated cells - % of apoptotic control cells)/(100 - % of apoptotic control cells). Recombinant EGF (rEGF) and EGFR inhibitor (AG1478) are from Sigma. The specific AhR antagonist alpha-naphthoflavone (alpha-NF, 10 μg/ml, Sigma) or agonist beta-naphthoflavone (beta-NF, 20 μg/ml, Sigma) were used for 1 h prior to PM2.5 exposure and/or apoptosis induction.
Cells were fixed 1 h by immersion at 4°C in 2.5% glutaraldehyde and 1% tannic acid in 0.1 M sodium cacodylate buffer, washed, postfixed in 2% osmium tetroxide deshydrated before embedding in Epon. Electron microscopy was performed with a transmission electron microscope (model Philips TECNAI 12), at 80 kV on ultrathin sections (60 nm).
Amphiregulin and GM-CSF secretion
Subconfluent 16HBE cells were exposed to PM2.5-AW for 4 h or 24 h and supernatants were recovered, centrifuged at 15,000 × g for 15 min at 4°C to pellet particles, and then frozen at -80°C until further analysis. The concentrations of Amphiregulin (AR) and GM-CSF released were evaluated with an enzyme-linked immunosorbent assay kit (ELISA, R&D Systems Europe; Abingdon, UK) according to the manufacturer's recommendations.
AhR gene silencing
16HBE cells were simultaneously seeded at 2 × 104 cells/cm2 either in T25 dishes (for Western Blot) or in a P24 well plate (for flow cytometry) and incubated under normal cell culture conditions overnight. Then, 10 nM of AhR siRNA (Hs_AHR_6 AlexaFluor-647, Ref SI03043971, Qiagen) or control non-silencing siRNA (AllStars Neg AlexaFluor-647, Ref 1027287, Qiagen) and HiPerFect Transfection Reagent (Qiagen) were mixed separately in medium and the formed-complexes were then added drop-wise onto the cells, according to the manufacturer's recommendations. At 48 h after transfection, the cells were subjected to our usual protocol: 4 h PM2.5 pretreatement and/or A23187 (3 μM) for additional 20 h.
Western Blots were performed according to the method previously described  and the primary antibodies used were: mouse-monoclonal anti-AhR (WH0000196M2, Sigma) and anti-Actin (A5441, Sigma). The secondary antibodies (peroxidase-conjugated) were anti-mouse immunoglobulin (A9044, Sigma). Immunoreactive bands were detected by chemiluminescence using a Chemiluminescent Sensitive HRP Substrate (BioFX Laboratories) using a FujiFilm LAS 4000 camera system.
All results are presented as the mean +/- standard deviation of three independent experiments. Data were analyzed using one-way ANOVA analysis of variance. The Dunnett's test was performed for all multiple comparisons versus control group. Moreover, the Student-Newman-Keuls test was used for all pairwise comparisons of mean responses among the different treatment groups (SigmaStat). Differences between groups were considered significant if the p value was less than 0.05.
This work was supported by Agence Nationale de la Recherche [0599-5 SET 024-01], Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot-Paris 7 (Bourse de Master, Melanie Leroux), Région Ile de France (Allocation post-doctorale, Ioana Ferecatu [F-08-1261/R]), ADEME-Primequal [0462C0056], CAMPLP (Caisse d'Assurance Maladie des Professions Liberales de Province, Paris, France), Renault (DIMAT, for the supply and chemical analysis of PM2.5 and PM organic extracts) and Legs Poix.
We thank Annie Jaeger and Marie Claude Gendron for their technical assistance and Emeline Assémat for advice on NHBE culture. We thank Mabel Jouve-San Roman from the Jacques Monod Institute (Paris, France) for help in electron microscopy. We also thank Sonja Boland, Martine Aggerbeck and Christophe Lemaire for their critical reading of the manuscript.
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