Research A Section 508–conformant HTML version of this article is available at https://doi.org/10.1289/EHP4178. Airway Hyperresponsiveness, Inflammation, and Pulmonary Emphysema in Rodent Models Designed to Mimic Exposure to Fuel Oil–Derived Volatile Organic Compounds Encountered during an Experimental Oil Spill Óscar Amor-Carro,1,2 Kathryn M. White,1 Rebeca Fraga-Iriso,1,2 Luis A. Mariñas-Pardo,1 Laura Núñez-Naveira,1 Beatriz Lema-Costa,1 Marta Villarnovo,1 Héctor Verea-Hernando,1 and David Ramos-Barbón1,2 1 Respiratory Research Unit, Complexo Hospitalario Universitario and the Instituto de Investigación Biomédica de A Coruña, A Coruña, Spain 2 Respiratory Department, Hospital de la Santa Creu i Sant Pau and the Biomedical Research Institute (IIb Sant Pau), Barcelona, Spain BACKGROUND: Fuel oil–derived volatile organic compounds (VOCs) inhalation is associated with accidental marine spills. After the Prestige petro- leum tanker sank off northern Spain in 2002 and the Deepwater Horizon oil rig catastrophe in 2009, subjects involved in environmental decontamina- tion showed signs of ongoing or residual lung disease up to 5 y after the exposure. OBJECTIVES: We aimed at investigating mechanisms driving persistent respiratory disease by developing an animal model of inhalational exposure to fuel oil–derived VOCs. METHODS: Female Wistar and Brown Norway (BN) rats and C57BL mice were exposed to VOCs produced from fuel oil mimicking the Prestige spill. Exposed animals inhaled the VOCs 2 h daily, 5 d per week, for 3 wk. Airway responsiveness to methacholine (MCh) was assessed, and bron- choalveolar lavage (BAL) and lung tissues were analyzed after the exposure and following a 2-wk washout. RESULTS: Consistent with data from human studies, both strains of rats that inhaled fuel oil–derived VOCs developed airway hyperresponsiveness that persisted after the washout period, in the absence of detectable inflammation in any lung compartment. Histopathology and quantitative morphol- ogy revealed the development of peripherally distributed pulmonary emphysema, which persisted after the washout period, associated with increased alveolar septal cell apoptosis, microvascular endothelial damage of the lung parenchyma, and inhibited expression of vascular endothelial growth fac- tor (VEGF). DISCUSSION: In this rat model, fuel oil VOCs inhalation elicited alveolar septal cell apoptosis, likely due to DNA damage. In turn, the development of a peculiar pulmonary emphysema pattern altered lung mechanics and caused persistent noninflammatory airway hyperresponsiveness. Such findings suggest to us that humans might also respond to VOCs through physiopathological pathways different from those chiefly involved in typical cigarette smoke–driven emphysema in chronic obstructive pulmonary disease (COPD). If so, this study could form the basis for a novel disease mechanism for lasting respiratory disease following inhalational exposure to catastrophic fuel oil spills. https://doi.org/10.1289/EHP4178 Introduction In 2002, the oil tanker Prestige split and sank approximately The health consequences of occupational or accidental inhalation of 200 km off the coast of Galicia in northwest Spain. As a result, oil-derived volatile organic compounds (VOCs) have been studied over 60,000 tons of class 2B fuel oil with high aromatic hydro- in various settings (Hauser et al. 1995; Koh et al. 2014; Raabe et al. carbon content were spilled in the ocean (Rodríguez-Trigo et al. 1998; Singhal et al. 2007; Uzma et al. 2008; Woodin et al. 1998). 2007). Dragged by currents and tides, successive floating layers Particularly, the exposure of cleanup workers and volunteers to oil of viscous fuel spread over large surfaces, affecting more than spills has raised special concern and achieved extensive media cov- 1,000 km of coast, mainly in Spain and also reaching Portugal and erage due to the repetitiveness of large environmental catastrophes France (Zock et al. 2007). This caused one of the most massive caused by petroleum tanker and marine oil rig disasters (Jernelöv environmental catastrophes before the Deepwater Horizon oil rig 2010; Zock et al. 2011). Studies on oil VOC exposure by cleaning spill in the Gulf of Mexico in 2009. During several months follow- personnel reported a variety of acute and short-term effects, includ- ing the Prestige spill, at least 300,000 persons, including volun- ing manifestations consistent with upper airway and whole respira- teers, fishers, military, and fuel company workers, inhaled VOCs tory tract irritation. Such data were collected from the Deepwater through their involvement in cleaning activities. Exposed subjects Horizon leak (Alexander et al. 2018; Goldstein et al. 2011) and other showed acute short-term symptoms including headache, dizziness, spills (Campbell et al. 1993, 1994; Janjua et al. 2006; Lyons et al. nausea, erythema, and upper airway irritation (Carrasco et al. 2006; 1999; Meo et al. 2008, 2009; Morita et al. 1999). Asthma attacks Rodríguez-Trigo et al. 2007; Suárez et al. 2005), as well as were also reported (Eggleston 2007; Goldstein et al. 2011; Leikauf increased DNA damage (Laffon et al. 2006). An extensive epide- 2002; Nurmatov et al. 2013). miological study was subsequently conducted in which the sub- jects’ respiratory symptoms, lung function, and markers of inflammation were analyzed. The data revealed evidence of Address correspondence to David Ramos-Barbón, Servei de Pneumologia, increased upper and lower respiratory tract symptom frequency Mòdul 2-4a Plta, Hospital de la Santa Creu i Sant Pau, Sant Antoni Maria (Zock et al. 2007), airway hyperresponsiveness (Rodriguez- Claret 167, 08025 Barcelona, Spain. Telephone: +34 93 556 5970. Fax: +34 Trigo et al. 2006; Zock et al. 2011) with no difference in baseline 93 556 5601. Email: dramosb@santpau.cat spirometry (Rodríguez-Trigo et al. 2010), and higher concentra- Supplemental Material is available online (https://doi.org/10.1289/EHP4178). tions of vascular endothelial growth factor (VEGF), basic fibro- The authors declare they have no actual or potential competing financial interests. blast growth factor (bFGF), and the oxidative stress marker 8- Received 16 July 2018; Revised 24 December 2019; Accepted 2 January isoprostane in exhaled breath condensate (Rodríguez-Trigo et al. 2020; Published 12 February 2020. 2010). Since 2 y elapsed from oil VOCs exposure to data collec- Note to readers with disabilities: EHP strives to ensure that all journal tion, the reports suggested late respiratory system effects. The content is accessible to all readers. However, some figures and Supplemental lower respiratory tract symptoms persisted in a subsequent sur- Material published in EHP articles may not conform to 508 standards due to vey at 5 y after the exposure (Zock et al. 2012). The concern over the complexity of the information being presented. If you need assistance accessing journal content, please contact ehponline@niehs.nih.gov. Our staff respiratory sequelae persisting after VOCs inhalation caused by will work with you to assess and meet your accessibility needs within 3 an environmental spill has been recently revisited through the working days. analysis of spirometry data from a large cohort exposed to the Environmental Health Perspectives 027003-1 128(2) February 2020 Deepwater Horizon leak. Among the spill response and cleanup had no clue a priori on the immunobiological pathways that workers, the studies found a higher risk of lung function decline might be involved, we tested the Wistar and BN rat strains, which at 1–3 y after the disaster in those subjects directly involved in have a constitutive immune deviation toward T helper 1 (Th1) decontamination procedures or recovery of oily plants, wildlife, and Th2 responses, respectively. The development of results or dead animals, and in those highly exposed to burning oil or gas from the rat strains and the C57/BL6 mouse brought us to limit (Gam et al. 2018a, 2018b). our experiments to the C57/BL6 strain in the case of the mouse. The data from the Prestige and the Deepwater Horizon stud- ies suggest the presence of late, persistent pathogenic mecha- Fuel Oil Source and Specifications nisms or unresolved lung damage underlying the abnormalities The Prestige fuel oil was classified number 6 as per the American observed. Such a hypothesis and the limitations of the field stud- Society for Testing and Materials (ASTM, http://www.astm.org/ ies to provide an insight into the disease mechanisms involved cgi-bin/resolver.cgi?D396-19a) and number 2 as per the French led us to develop a rodent model of oil-derived VOC inhalation National Organization for Standardization (AFNOR, https:// mimicking exposure to the Prestige spill. Data were collected at www.boutique.afnor.org/standard/m15-011/liquid-mineral-fuels- two time points: upon the termination of exposure and following properties-of-heavy-fuel-oil-no-2/article/727394/fa018130) a washout period, to reflect, respectively, early and late effects on (Rodríguez-Trigo et al. 2007). A heavy fuel oil blend resembling pulmonary function, inflammation, and lung structure. the spill released by the Prestige tanker was synthesized and gen- erously provided by Repsol (Repsol Technology Center). Table Methods S1 summarizes the fuel oil parameters. The study protocol and experimental procedures were approved by a Government Animal Ethics Committee (Comité Etico  de Exposure System Experimentación Animal of Xunta de Galicia) in compliance The animals were exposed to fuel oil vapor inhalation using a with European Union Directive 86/609 (https://op.europa.eu/en/ flow-through exposure tower [Electro-Medical Measurement publication-detail/-/publication/cc3a8ccb-5a30-4b6e-8da8-b13348 Systems (EMMS)]. This equipment was specially adapted for this caeb0c/language-en) and Royal Decree 1201/2005 of Spain project by EMMS engineers (Figure S1) in consultation with the (https://www.boe.es/eli/es/rd/2005/10/10/1201). Animal handling, British Toxicology Society. An inhalation tower designed for ho- experimental procedures, and data collection and reporting were mogeneous gas distribution was fed a constant airflow that passed carried out in compliance with the Animal Research: Reporting through a fuel oil–vaporizing chamber and was delivered through- In Vivo Experiment (ARRIVE) guidelines (Kilkenny et al. out the tower to nose-only exposure ports. Setup testing showed 2010). no oxygen depletion at the exposure ports. For each 2-h exposure session, 2 mL of fuel oil was poured into a vapor generator Animals immersed in a 60°C water bath to decrease oil viscosity and Female Wistar and Brown Norway (BN) rats and female C57BL/6 improve vapor release. The constant airflow was maintained mice were purchased from Harlan Interfauna Ibérica and housed in throughout the circuit by balance-controlled positive and negative polycarbonate cages (four to five mice or two to three rats per cage) air pumps. Air pressure was slightly negative at the exposure ports assembled within a high-efficiency particulate air (HEPA)–filtered to ensure that the animals were only inspiring air as demanded by ventilated rack under controlled temperature (21–24°C) and hu- spontaneous tidal respiration and were not breathing forcefully. midity (40–55%), with a 12/12-h light/dark cycle and ad libitum The fuel oil–contaminated air was filtered through a series of char- access to food (Harlan Interfauna Ibérica) and water. Rats and mice coal filters and dehumidified through silica gel filters before it was were purchased with a specified 6-wk age upon delivery, and a 7-d released into a certified chemicals exhaust hood hosting the adaptation period followed animal supply before carrying out any equipment. experimental procedures. At the time point of pulmonary function testing, the animals were 10–13 wk old, with the rats weighing Fuel Oil Volatile Organic Compound Composition 150–180 g and the mice 18–23 g. Pulmonary function testing was Benzene, toluene, ethylbenzene, and xylene (BTEX) contents were normalized on a per-animal weight basis recorded at the moment determined at the inhalation ports to quantitatively assess VOCs of anesthesia induction. in the air inhaled by the animals. BTEX was measured using PerkinElmer thermal desorption tubes (Sigma-Aldrich) and an air Study Design sampling pump set to sample at 60:08-mL=min flow for 2 h Each set of experiments comprised a group of animals exposed to fuel in sequential 30-min samples that were analyzed separately. The oil vapors during 2-h sessions, 5 times per week, for 3 wk. Additional samples were eluted by thermal desorption TurboMatrix ATD-400 groups underwent the same exposure schedule and were then left (TurboMatrix; PerkinElmer) into an SGE-BPX capillary column unexposed, breathing HEPA-filtered air only, for a 2-wk washout pe- (Grace) and analyzed by gas chromatography coupled to an ion trap riod before data collection. The latter was aimed at capturing lasting gas spectrometer (Polaris Q; Thermo Finnigan). The experimental effects, such as those reported from human subjects after an elapsed fuel oil–derived VOCs peaked at 30 min and decreased over time. time following the Prestige fuel oil exposure (Rodríguez-Trigo et al. Table 1 summarizes the BTEX measurements. VOCs composition 2010; Zock et al. 2007, 2012). Nonexposed animals breathing comprised the BTEX compounds reported in the field measurements HEPA-filtered air acted as control groups. The control, exposed, and performed by the Spanish government research agency Consejo exposed with postexposure washout groups are hereinafter referred to Superior de Investigaciones Científicas (CSIC) during the coast as “Control,” “Exposed,” and “Rested,” respectively, for all figures cleaning labors that followed the Prestige catastrophe (CSIC 2003, and data tables. All experimental groups were n = 6, and the animals 2005). However, direct comparisons of the proportions of each were randomly assigned to each experimental group. BTEX compound are limited by several factors. In the CSIC field Because airway responsiveness to methacholine (MCh) chal- measurements, the BTEX content was analyzed as milligrams per lenge was increased in the exposed human subjects, our starting kilogram in the transported fuel oil, the marine residues, and the hypothesis on the underlying pathophysiology was oriented at an coastal deposits, but not as its concentration in the ambient air where immune-driven response eliciting airway inflammation. Since we the cleaning crews were working. Also, the volatilization kinetics of Environmental Health Perspectives 027003-2 128(2) February 2020 Table 1. Volatile organic compounds (VOCs) analysis at inhalation ports. Time (min) Benzene (ng) Toluene (ng) Ethylbenzene (ng) m- + p-Xylene (ng) o-Xylene (ng) 0–30 17.2 1,122.0 344.0 643.0 661.0 30–60 12.4 503.0 31.3 64.0 53.0 60–90 12.4 602.0 24.3 51.7 38.8 90–120 7.9 448.0 6.9 10.6 6.4 Total (ng) 49.9 2,675.0 406.5 769.3 759.2 Air concentration (lg=m3 ) 6.92 371.03 56.38 106.70 105.30 Note: Benzene, toluene, ethylbenzene, and meta-, para-, ortho-xylene isomers (BTEX) gas chromatography analysis. A 2-mL fuel oil aliquot was delivered into the vaporization cham- ber over 2 h as for the animal exposure experiments. Air samples were collected at the inhalation ports of the exposure tower during the time intervals indicated. For each BTEX com- pound, the mass collected during each time interval and its total for the entire period are expressed in nanograms (ng). Whole air concentration is calculated from the total mass divided by the total sampled volume and expressed as micrograms per cubic meter (lg=m3 ). the various BTEX compounds differ, and the fuel oil reaching testing kit materials generously given by eBioscience; assay lim- extensive coastal areas did not preserve full stability in its composi- ited to the BN rat). tion. Considering such limitations to correlate the BTEX air con- centrations in our model with the BTEX relative masses in the Lung Tissue Processing field-sampled fuel oil, our data suggest a higher release of toluene Immediately after BAL collection, the pulmonary vascular circuit by the fuel oil employed in our model. Due to the impossibility of was washed with 2 mM ethylenediaminetetraacetic acid (EDTA) in obtaining samples of the original fuel oil as released by the PBS, injected through the right ventricle, and flushed out through an Prestige tanker, synthesizing the experimental fuel oil for this opening in the abdominal aorta. The cardiopulmonary block was work was considered a best feasible approach to model the environ- then dissected out and connected to a formalin recirculation system mental exposure. through the tracheal cannula. The lungs were thus inflated and fixed with 10% formalin at 25 cmH2 O intratracheal standard pres- Airway Hyperresponsiveness to Methacholine sure for 22 h. From the inflated, fixed lungs, sagittal slices were To study the effects of fuel oil vapor inhalation on lung function, cut with a scalpel, placed into standard tissue processor cassettes airway hyperresponsiveness to MCh was evaluated 3 d after the (ThermoFisher Scientific), and paraffin embedded with the lung last exposure or following the 2-wk washout period. FlexiVent® midsagittal plane facing the microtome cutting side as per previ- equipment (SCIREQ) was employed for rodent mechanical venti- ously established methodology for airway and parenchyma quan- lation and pulmonary function testing by forced oscillation tech- titative morphology (Guerassimov et al. 2004; Ramos-Barbón nique using a linear first-order single-compartment model. The et al. 2005). For this purpose, a 2-mm-thick lateral slice was animals were anesthetized with 4% inhaled sevoflurane (Sevorane®; trimmed and discarded, and the slice generated by sectioning at Abbott Laboratories) and subjected to tracheotomy with standar- the lung midsagittal plane was processed for paraffin embedding. dized tracheal cannulae (EMMS). Neuromuscular blockade was From the paraffin blocks, 5-lm-thick microtome tissue sections induced with 1 mg=kg of intraperitoneal rocuronium bromide were stained with hematoxylin and eosin (H&E) for general his- (Organon Pharmaceuticals). Mechanical ventilation was set topathological assessment, periodic acid–Schiff (PAS) to quan- (mice/rats, respectively) at 250/90 breaths per minute with a tidal tify goblet cells and airway mucus load, and Masson’s trichrome volume of 0:14=1:5 mL and 2=3 cmH2 O positive end-expiratory to evaluate airway subepithelial fibrosis. pressure (PEEP). Following baseline measurements, the animals were administered doubling doses of aerosolized MCh (Sigma- Quantification of Pulmonary Emphysema Aldrich) (Figure 1) with 25 lL of each solution delivered into an Two quantitative morphology methods were employed to measure Aeroneb ultrasonic nebulizer (Aeroneb® Lab Nebuliser, Buxco pulmonary emphysema on H&E-stained sections. Two midsagittal Research Systems) connected to the inspiratory arm of the slides per animal, from the right and left lung, respectively, were FlexiVent® ventilator. Following each MCh dose, pulmonary re- examined. The lung parenchyma was thoroughly sampled into sistance (RL ) was measured every 15 s for 5 min to capture the images that were black/white binarized to discern the alveolar wall postdose RL peak. tissues from airspaces, and the surface area of the digitally extracted parenchymal tissue was referenced to the sampled surface area to Bronchoalveolar Lavage Collection and Analysis express parenchymal tissue density as a dimensionless percentual index. For the alternative method, we calculated the parenchymal Following pulmonary function recordings, the animals were eu- mean linear intercept (MLI), a well-established parameter for the thanized under deep anesthesia by intravenous injection of quantitative assessment of pulmonary emphysema on tissue sec- 0:3 mL of 7.45% potassium chloride (Braun). Bronchoalveolar la- tions (Dunnill 1962; Parameswaran et al. 2006; Robbesom et al. vage (BAL) was immediately harvested through the tracheal tube 2003). For this purpose, 15 images were randomly sampled at by use of 5 mL of saline in 1-mL fractions for the mice and 200 × magnification from subpleural regions, and a grid with 6 25 mL in 5-mL fractions for the rats. BAL was centrifuged at equally distributed horizontal lines and 8 vertical lines was overlaid 500 × g for 10 min. The cellular pellets from the BAL fractions (Figure S2). The line intercepts with alveolar walls were counted, were pooled and resuspended in 1 mL phosphate-buffered saline and MLI was calculated as: (PBS), and total live leukocytes were counted in a Neubauer he- macytometer (Marienfeld) using trypan blue dye (ThermoFisher V × Lv + H × Lh Scientific) exclusion. Interleukin 1 alpha (IL-1a), macrophage MLI = , Ni chemotactic protein-1 (MCP-1), tumor necrosis factor-a (TNFa), interferon gamma (IFN-c), granulocyte–monocyte colony stimu- where V is the number of vertical lines, Lv is the length of each ver- lation factor (GM-CSF), and IL-4 concentrations were analyzed tical line, H is the number of horizontal lines, Lh is the length of in frozen, preserved (−80 C) BAL supernatants by flow cytome- each horizontal line, and Ni is the sum of all intercepts. A BX61/ try using a bead-based multiplex immunoassay (FlowCytomix™; BX62 Olympus microscope equipped with a DP70 Olympus Environmental Health Perspectives 027003-3 128(2) February 2020 septal cells was calculated by stereology (Hsia et al. 2010), analyz- ing 10 random fields per animal on 20-lm-thick tissue sections and discarding the cells whose nuclei were in contact with the focal plane corresponding to the microscope slide surface. By this proce- dure, TUNEL+ alveolar septal cells were counted in the alveolar walls, normalized by cubic millimeter of parenchymal tissue, and expressed as apoptotic cells per cubic millimeter. The reference parenchymal tissue volume was quantitated by digital extraction as for the calculation of pulmonary emphysema. The microscope sample fields were captured with a 40 × bright-field objective and yielded a 152,053:08-lm2 surface area and 3,041,061:69-lm3 vol- ume upon calibration. Field sampling from one midsagittal right lung microscopy slide per animal yielded appropriate statistical power for the effects detected in the TUNEL-processed specimens and in the CD31, VEGF, CD8, and neutrophil elastase–immuno- stained preparations (next section). Immunohistochemical Detection and Quantification of CD31, Vascular Endothelial Growth Factor, CD8, and Neutrophil Elastase Lung tissue sections were deparaffinized and rehydrated through 5- min baths in xylene (three baths), 100% ethanol (two baths), and 95% and 70% ethanol, and placed in PBS or Tris-buffered saline. High-temperature epitope unmasking was done in Universal Antigen Retrieval Reagent (Abcam) using a 2100 Retriever (Aptum Biologics Ltd.). The tissue sections were then permeabilized in 0.2% Triton-X 100 (Sigma-Aldrich) and blocked with 2.5% goat serum or horse serum (Vector Laboratories) as appropriate. The following pri- mary antibodies (all from Abcam) were used: anti-CD31 rabbit monoclonal EPR17259 at 1:500 dilution (1:2 lg=mL). Anti-VEGF rabbit polyclonal (ab53465) at 1:300 dilution (3:3 lg=mL), anti- CD8 mouse monoclonal OX-8 at 1:200 dilution (5 lg=mL), and anti-neutrophil elastase rabbit polyclonal (ab21595) at 1:200 dilution (5 lg=mL). CD31 immunostaining was selected as a procedure to label endothelial cells, allowing for precise detection of microvascu- lature down to the capillary level (Pusztaszeri et al. 2006). For CD31 and neutrophil elastase immunostaining, endogenous peroxidase was blocked with BLOXALL® solution (Vector Laboratories), and a goat anti-rabbit IgG secondary antibody with polymerized horserad- ish peroxidase (ImmPRESS® reagent; Vector Laboratories) was Figure 1. Airway responsiveness to methacholine challenge. The plots repre- applied. CD31 detection was developed with Vector Novared sent the pulmonary resistance (RL ) peak after each methacholine (MCh) dose. The Exposed animals inhaled fuel oil–derived VOCs for 2-h sessions, 5 times chromogen, and neutrophil elastase with diaminobenzidine (DAB)– per week, for 3 wk. The Rested animals were subjected to the same VOC ex- nickel (Vector Laboratories) on respective slide sets. The anti-VEGF posure regime followed by a 2-wk washout period. The Control (CTRL) ani- and anti-CD8 primary antibodies were followed by a) biotinylated mals had no exposure. These group definitions apply uniformly to the Wistar goat anti-rabbit IgG and horse anti-mouse IgG secondary antibodies, and Brown Norway rats and the C57BL/6 mice for all subsequent figures and respectively (both from Vector Laboratories) at 1:200 dilution data tables as applicable. RL values of (A) Wistar rats, (B) Brown Norway (7:5 lg=mL); b) an alkaline phosphatase avidin–biotin complex rats, and (C) C57BL/6 mice are shown. Error bars represent the standard error of the mean. *p < 0:05 vs. CTRL for both the Exposed and Rested groups. (ABC-AP, Vector Laboratories); and c) Vector® Red chromogen Statistical analysis: one-way analysis of variance (ANOVA) with Fisher’s (Vector Laboratories). All second-step reagents (ImmPRESS® rea- least significant difference (LSD) test for pairwise post-ANOVA comparisons gent and biotinylated secondary antibodies) were adsorbed in 10% at each MCh concentration level; n = 6 for all experimental groups. normal rat serum (STEMCELL Technologies). Chromogen devel- opment was followed by hematoxylin QS counterstain (Vector Laboratories), dehydration through two 100% ethanol and three xy- camera was used for image capture, and ImageJ software (version lene baths, and permanent mounting with VectaMount medium 1.47; National Institutes of Health) was used for digital image (Vector Laboratories). processing and quantitative procedures. The volume density (dimensionless) of CD31-immunostained endothelium within the alveolar walls was calculated by digital signal thresholding and extraction and referenced to the digitally Detection and Quantification of Apoptotic Structural extracted parenchymal tissue, similar to the procedure referred Alveolar Septal Cells for the calculation of parenchymal volume density in the quanti- Apoptotic cells were detected in lung parenchyma by the termi- fication of emphysema, but using a 60 × oil immersion objective nal deoxynucleotidyl transferase dUTP nick end labeling for image capture in this case. The numerical density of VEGF+ (TUNEL) assay using ApopTag® kits (Millipore) per the manu- and CD8+ cells was calculated by cell counting normalized by facturer’s instructions. The numerical density of apoptotic alveolar cubic millimeter of parenchymal tissue, similar to the numerical Environmental Health Perspectives 027003-4 128(2) February 2020 density of apoptotic alveolar septal cells. All calculations were Bronchoalveolar Lavage Analyses and Airway also done with ImageJ software (version 1.47). Histopathology Total BAL cell counts (Figure 2; numerical data and p-values in Table S3) did not show any significant differences among the ex- Data Analysis perimental groups for any of the rat strains or the C57BL mouse. Data distributions are represented by the mean and standard error. The BN rat showed a tendency toward a progressive increase in Statistical significance was analyzed by one-way analysis of var- total BAL cells through the VOCs inhalation and washout peri- iance (ANOVA) followed by post hoc Fisher’s least significant ods, which did not reach statistical significance (p = 0:22 in the difference test or Games-Howell test for unequal variances. Exposed group and p = 0:09 in the Rested group vs. the Control Student’s t-test was used where appropriate for two-group inde- animals). Since this trend in the BN rat was close to significance pendent comparisons. Inferences on the size of effects are in the Rested group, we performed a multiplex cytokine analysis reported as mean increment [mean difference (95% confidence in the BAL fluid of this strain, which did not show any significant interval of the mean difference)]. A p-value of less than 0.05 was difference supportive of inflammatory activity in the Exposed or considered statistically significant. SPSS statistical software was Rested groups. The cytokine analysis battery included IL-1a, employed (version 16.0; IBM Corp.). MCP-1, TNFa, IFN-c, GM-CSF, and IL-4 (Figure 2D). This analysis, performed with a test kit sample, was limited to the BN rat, selected based on its BAL leukocyte counts profile. Because Results of the spread variability of the cytokine distributions obtained and its null outcome, we estimated a very low probability of Evaluation of Airway Responsiveness to Methacholine obtaining any informative data from the Wistar rat and the Challenge C57BL mouse, and we deemed it not cost-effective to pursue Wistar and BN rats and the C57BL mice were subjected to RL such analyses. determinations upon MCh challenge in vivo for the Control, A systematic scan of the H&E-stained tissue sections did not Exposed, and Rested groups, respectively, as specified in the reveal any pathological findings in the conducting airways. Notably, study design. RL curves are plotted in Figure 1, and the corre- no airway inflammation or remodeling were found. Representative sponding numerical data and exact p-values are shown in Table sections of intrapulmonary airways are shown in Figure 3, where it S2. Both the Wistar and BN rats showed airway hyperresponsive- can be seen that VOCs inhalation did not lead to any detectable ness to MCh in the exposed groups, defined as per significant dif- inflammatory infiltrates or signs of airway remodeling as compared ferences in RL at the two highest MCh doses (p < 0:05 over the with controls in the BN rats (Figure 3A–C), Wistar rats (Figure 3D– Control group for the 12.5 and 25 mg=mL MCh doses, respec- F), or C57BL/6 mice (Figure 3G–I). PAS staining did not reveal any tively; Figure 1A,B). The RL curves were similar for both rat alterations of mucus-producing cells or overall epithelial mucous strains in the Rested groups, which reflected that airway load, and we did not observe differences in extracellular matrix depo- hyperresponsiveness persisted despite discontinuing VOC expo- sition or abnormal patterns such as subepithelial fibrosis in the sure. Conversely, no changes in respiratory parameters were Masson’s trichrome–stained specimens (Figure 4). Overall, the anal- found in the C57/BL6 mice at any data cutoff (Figure 1C). yses on the cellular and fluid BAL fractions and the histopathological Figure 2. Bronchoalveolar lavage (BAL) analysis. Total BAL leukocytes were counted at the end of VOCs exposure (Exposed group) and after a 2-wk wash- out (Rested group) vs. Control animals (CTRL). BAL leukocyte counts are shown for (A) the Wistar rat, (B) Brown Norway rat, and (C) C57BL/6 mouse. (D) BAL cytokine concentrations measured in the Brown Norway rat groups by a bead-based multiplex flow cytometry immunoassay. Minimum detectable levels were 8:5 pg=mL for interleukin 1 alpha (IL-1a), 0:5 pg=mL for macrophage chemotactic protein 1 (MCP-1), 4:3 pg=mL for tumor necrosis factor-a (TNFa), 8:3 pg=mL for interferon gamma (IFN-c), 5:0 pg=mL for granulocyte–monocyte colony stimulation factor (GM-CSF), and 0:3 pg=mL for IL-4. Graph bars rep- resent mean and standard error of the mean in all plots. One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test for pair- wise post-ANOVA comparisons; n = 6 for all experimental groups. Environmental Health Perspectives 027003-5 128(2) February 2020 Detection and Quantitative Analysis of Pulmonary Emphysema On histopathological examination, the Wistar and BN rats showed pulmonary emphysema that predominantly affected the most pe- ripheral parenchymal regions in a subpleural location in both the Exposed and Rested groups (Figure 5A–F). Quantitative morphol- ogy by the digital extraction and MLI methods jointly showed a progression whereby the parenchymal tissue density was signifi- cantly decreased at the end point of exposure, and further tissue destruction occurred during the washout period (Figure 5G–J; quantitative data and p-values given in Tables S4 and S5). The C57BL/6 mice did not show any changes in the lung paren- chyma on histopathological examination, whether upon exposure or after the washout, compared to the Control group (Figure S3). Similar to the airways, a thorough scan of the lung paren- chyma did not reveal any inflammatory infiltrates. To reassure Figure 3. Airway histopathology in hematoxylin and eosin–stained tissue the absence of neutrophil involvement associated with the devel- sections. Micrographs show representative cross-sectioned airways from opment of emphysema, we immunostained neutrophil elastase in the respective strains and experimental groups as indicated. Scale bars: the rat lung specimens. Figure S4 shows examples of circulating 200 lm. neutrophils as an internal staining control. We found such neutro- phils in limited blood remnants that were retained in some vessels despite the pulmonary vascular circuit wash performed during examination of stained tissue sections ruled out any detectable signs dissection. All experimental groups were devoid of extravascular of persistent airway inflammation and remodeling underlying the neutrophils infiltrating any tissue structures. sustained airway hyperresponsiveness. The experimental design did not aim at detecting possible acute lung inflammation during early VOC exposure. Detection and Quantification of Structural Alveolar Septal Cell Apoptosis In search of a mechanism underlying the development of pulmo- nary emphysema, we detected and quantified alveolar septal cell apoptosis in the rats. Both the Wistar and BN rat strains showed a significant increase in the frequency of TUNEL+ alveolar septal cells in the Exposed groups vs. the respective Control groups, and this effect was maintained after the washout period in the Rested groups (Figure 6B,C and Figure 7; Table S6). Frequency of CD8+ T Cells in the Lung Parenchyma To explore a possible role of CD8+ T cells as potential inducers of alveolar septal cell apoptosis through cytotoxicity, we quanti- fied the numerical density of immunostained CD8+ cells in the parenchyma (Figure 8; numerical data and p-values given in Table S7). BN control rats had a higher baseline frequency of CD8+ cells compared with Wistar rats. VOCs inhalation did not lead to consistent changes in the Exposed groups, but there was a significant decrease of CD8+ cell numerical density in the Rested groups of both strains. Analysis of Parenchymal Microvascular Response To analyze whether the development of emphysema was associated with alterations of the parenchymal microvasculature, we labeled the endothelium by CD31 immunostaining. The general CD31 im- munostaining pattern on different lung structures is shown in Figure S5. Consistent with previous reports (Pusztaszeri et al. 2006), the CD31 signal accurately delineated the parenchymal capillary net- work and allowed us to quantify its volume density within the alveo- lar walls. Upon overall microscopy examination, we observed a Figure 4. Periodic acid–Schiff (PAS) and Masson’s trichrome staining. noticeable difference in CD31 signal density between the subpleural Lung tissue sections were stained with PAS to identify goblet cells and eval- regions and the rest of the lung parenchyma in the animals of the uate the overall airway epithelial mucus load. Masson’s trichrome was Exposed and Rested groups. This finding prompted us to acquire employed to evaluate extracellular matrix deposition. The panels show repre- two separate sampling sets for quantitative morphology: central and sentative cross-sectioned airways for the respective stainings, animal strains, and experimental groups as indicated. No pathological alterations were iden- subpleural parenchyma (Figure 9; numerical data and p-values tified in terms of goblet cell hyperplasia or hypertrophy, overall epithelial given in Table S8). The CD31+ endothelium volume density was mucus load, or subepithelial fibrosis as a result of volatile organic com- homogeneous across the parenchymal regions in the Control ani- pounds (VOCs) inhalation. Scale bar: 100 lm. mals within the Wistar and BN strains. The BN control rats showed Environmental Health Perspectives 027003-6 128(2) February 2020 Figure 5. Effect of VOCs inhalation on lung parenchyma. The micrographs (A–F) show hematoxylin and eosin–stained lung sections capturing the subpleural region of lung parenchyma. All tissue sections are oriented showing the pleura on top. The panels correspond to the Wistar and Brown Norway rats and the re- spective experimental groups, as indicated. Pulmonary emphysema is seen as a decreased number of alveolar walls due to tissue destruction. The bar graphs (G–J) represent mean and standard error values for the corresponding quantitative morphology measurements by digital parenchymal extraction (G–H) and mean linear intercept (MLI) method (I–J) for the Wistar rat (G,I) and Brown Norway rat (H,J), respectively. Scale bar: 200 lm. *p < 0:05 vs. Control (CTRL) group. †p < 0:05 vs. both Control and Exposed groups. One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test for pair- wise post-ANOVA comparisons; n = 6 for all experimental groups. a tendency to lesser CD31 density than their Wistar counterparts, quantified the numerical density of VEGF+ cells in the alveolar which reached significance in the subpleural region (p = 0:029), at- walls (Figure 10; numerical data and p-values given in Table S9). tributable to a constitutive interstrain difference. In the Exposed Similar to CD31, there was an interstrain difference where the con- groups, the fuel oil VOCs inhalation led to a significant decrease trol BN rats had a significantly lower VEGF expression profile that of the CD31 volume density in both the central and subpleural the control Wistar rats (p < 0:001). Since the immunostaining parenchyma, this effect being drastic in the latter region. This batches comprised a balanced presence of slides from all experi- outcome was comparable between both rat strains. In the Wistar mental groups of both rat strains together, and the timing of reagent rat, the Rested group showed a significant recovery of CD31 vol- incubations, including chromogen development, was sequenced to ume density within the remnant alveolar walls of the subpleural be strictly systematic on a per-slide basis, we deem the observed region, which morphologically generated a peculiar budding interstrain differences not attributable to interference related to sprout aspect. The Rested BN rats did not show such significant batch staining. In both strains, the Exposed groups had a significant, recovery. strong inhibition of VEGF expression that remained flat in the The findings from the CD31-labeled microvasculature analysis Rested groups. and the reported increase of VEGF in exhaled breath condensate from subjects involved in the Prestige spill decontamination (Rodríguez-Trigo et al. 2010) led us to immunostain VEGF. As in Discussion previously reported lung VEGF expression profiling (Berse et al. The field research conducted after the Prestige tanker and the 1992; Medford et al. 2009), we found the VEGF signal predomi- Deepwater Horizon oil rig disasters left a knowledge gap about nantly located in alveolar septal cells and alveolar macrophages. We the pathophysiological processes that may have underlain the Environmental Health Perspectives 027003-7 128(2) February 2020 Figure 7. Distribution of apoptotic alveolar septal cells. For quantitative mor- phology, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL+ ) alveolar septal cells were identified through high-resolution field sampling. The images shown here are low-magnification micrographs to illus- trate the overall frequency and distribution of apoptotic alveolar septal cells in wide microscopy fields. The micrographs represent the Wistar and Brown Norway rat strains and the respective experimental groups, as indicated. For each rat strain and experimental group, the upper image is a direct micrograph, and the lower image is a grayscale replica where the location of apoptotic cells, as per identification at high magnification, is pinpointed (red arrows). Scale bar: 50 lm. actual Prestige spill at the human exposure zones were performed by a gas spectrometry analytics facility. Ultimately, the data pro- Figure 6. Detection of apoptotic alveolar septal cells in rat lung parenchyma. duction procedures were done by a respiratory research unit Apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP branched into two hospital research institute sites. Laboratory nick end labeling (TUNEL) technique on lung tissue sections. (A) Detail of modeling of an environmental catastrophe implies limitations, and an apoptotic alveolar septal cell, likely a type I pneumocyte, identified by its some assumptions are unavoidable. We analyzed BTEX concen- purple-stained nucleus with condensed chromatin. The bar graphs show the quantitative morphology data on the frequency of alveolar septal TUNEL+ trations in the air from the exposure tower ports to provide our best cells for the Control, Exposed, and Rested experimental groups in (B) the accuracy on what precisely the animals were breathing, yet the Wistar rat, and (C) the Brown Norway rat. Scale bar: 10 lm. *p < 0:05 vs. comparability of these data with the field fuel oil analyses is limited Control (CTRL) group. One-way analysis of variance (ANOVA) with by several factors as detailed in the “Methods” section. The extent Fisher’s least significant difference (LSD) test for pairwise post-ANOVA to which the VOCs release in this model reflects the human expo- comparisons; n = 6 for all experimental groups. sure mostly relies on the customized synthesis of the model fuel oil based on the Prestige payload specifications. Another limitation to extrapolate our data to the human experience is the inability to ana- lasting respiratory effects, observed years after the respective lyze any gender-related differences, since only female mice and spills, in the catastrophe response crews exposed to VOCs inhala- rats were employed in the experiments. Since the primary outcome tion (Gam et al. 2018a, 2018b; Rodríguez-Trigo et al. 2010; Zock that we aimed to reproduce was airway hyperresponsiveness, and et al. 2011, 2007). Here, we aimed at a mechanistic insight on re- our a priori expectations were on airway inflammatory disease, we spiratory system damage by developing an experimental model of opted for employing female animals as often done in asthma mod- fuel oil–derived VOCs inhalation mimicking the Prestige catastro- els. The overall asthma prevalence and mortality are higher in phe. The system setup involved interdisciplinary collaboration women (Akinbami et al. 2012; Moorman et al. 2012), and this epi- among various entities to achieve challenging technical goals. The demiological observation may have historically tilted rodent study fuel oil was blended at a research refinery facility based on asthma models towards the frequent use of female animals, yet known parameters from the Prestige tanker payload. The ad hoc without generating much evidence on possible gender-related dif- production of a VOCs-generating and decontamination exhaust ferences in the outcomes of such models. More recent comparative system and its coupling to a standardized rodent inhalation tower studies in murine asthma reported significantly higher allergen- involved research, manufacturing, and testing by a specialized en- specific IgE, Th2 cytokines, eosinophilic inflammation, and airway gineering firm. VOC sampling and profiling at the equipment inha- hyperresponsiveness in the female animals (Blacquière et al. 2010; lation ports and data matching with field measurements from the Takeda et al. 2013). Finally, the sequence of procedures performed Environmental Health Perspectives 027003-8 128(2) February 2020 Figure 8. Quantification of CD8+ T cells in lung parenchyma. CD8+ cells were immunostained (red signal) and their numerical density quantified in the alveo- lar walls. Counterstain: hematoxylin QS. (A) Example of a high-magnification field as employed for quantitative morphology. The micrograph corresponds to a control Wistar rat. (B) Lower-magnification capture encompassing a large parenchyma field, where a number of CD8+ cells can be identified. The image, from another control Wistar rat, is representative to provide a visual sense of the frequency of CD8+ T cells in control animals. (C) Example from the Wistar Rested group showing a wide field under the same magnification as in (B). A number of fields sampled from the Rested groups of both rat strains did not con- tain any CD8+ cells. The image shown here has intentionally captured one CD8+ cell as a reference, visible in the upper-left quadrant. (D) Numerical density of CD8+ T cells in the alveolar walls. *p < 0:05 vs. Control (CTRL). †p < 0:05 vs. Exposed. Scale bar: 50 lm in (A); 100 lm in (B,C). One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test for pairwise post-ANOVA comparisons; n = 6 for all experimental groups. ‡p < 0:05 vs. Wistar for independent interstrain group comparison (Student’s t-test). in the same animals may have involved some confounding risk due but only limited comparative work across species has been pub- to carryover effects. This uncertainty particularly applies to the lished, with a homogeneous response not always obtained. The rat obtention of biological specimens after ending pulmonary function has been reported to be more sensitive than the mouse to low- testing with MCh challenges under invasive mechanical ventila- respiratory-tract effects of 1,6-hexamethylene diisocyanate inhala- tion. Although such a methodological sequence is often performed tion (Pauluhn 2008). Anatomical and physiological differences exist in experimental asthma models and has been in extended use in the respiratory tract and pulmonary vasculature between the (Ramos-Barbón et al. 2004), a lack of any effects of the in vivo mouse and rat, which may introduce complexity factors for respira- invasive procedures on the biological samples collected subse- tory disease modeling. In our study, given that both rat strains quently is not warranted. yielded a comparable output for the relevant outcomes detected, we To explore the suitability of rodent species to model the respira- did not pursue further investigations across different mouse strains tory effects recorded in humans, we performed experiments in mice following the null response of the C57BL/6. and rats. The single mouse strain tested, C57BL/6, failed to repre- Both Wistar and BN rats showed airway hyperresponsiveness at sent in vivo the primary functional outcome obtained from humans, the end of the exposure period, which was maintained without signif- airway hyperresponsiveness to MCh challenge, whereas both rat icant variation after the washout. This finding suggests a persistent strains, Wistar and BN, did reproduce such an outcome comparably. disease mechanism regardless of the exposure washout, which is The mouse has been extensively employed for inhalational studies, also independent from the Th1 vs. Th2 constitutive immunological Environmental Health Perspectives 027003-9 128(2) February 2020 Figure 9. Immunostaining and quantitative morphology of parenchymal microvasculature. Lung endothelium was labeled by CD31 immunostaining, and its volume density (V/V, dimensionless) in the alveolar walls was quantified. Due to noticeable differences in the CD31 signal density between peripheral (sub- pleural) and central parenchymal regions, two separate sampling sets were acquired and analyzed for quantitative morphology, respectively. (A) Representative images of CD31-immunostained (brown signal) parenchyma from the central and subpleural regions for the Control, Exposed, and Rested groups of the Wistar and Brown Norway rat strains, as indicated. Counterstain: hematoxylin QS. The arrow in the left-hand image of the Wistar subpleural set indicates the visceral pleura. To facilitate image interpretation, the pleura was captured in the approximate same position in all subpleural images. The scale bar (50 lm) in the upper-left panel applies to all micrographs. (B) Quantitative morphology of CD31-immunostained endothelium V/V for the experimental groups, parenchymal regions, and rat strains, as indicated. *p < 0:05 vs. Control. †p < 0:05 vs. Exposed. ‡p < 0:05 vs. central parenchyma, intragroup. One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test for pairwise post-ANOVA comparisons; n = 6 for all experimental groups. §p < 0:05 vs. Wistar for independent interstrain group comparison (Student’s t-test). background of the strains. Fuel oil exposure did not lead to any sig- response by acting as haptens through carrier protein binding as nificant changes in airway histopathology. A thorough scan of the reported for occupational exposure to toluene, one of the VOCs H&E-stained and neutrophil elastase–immunostained tissue sec- released by the fuel oil blend employed in our study (Karol 1983). tions did not detect any traces of airway inflammatory infiltrates In the absence of an adaptive immune response, VOCs may inter- nor signs of structural remodeling of the airway wall. PAS staining act with innate immunity pathways through contact with the air- did not reveal goblet cell hypertrophy or hyperplasia nor increased way epithelium and elicit inflammation, as described for reactive mucus production, and the Masson’s trichrome–stained specimens airways dysfunction (Vincent et al. 2017). Given the various pos- did not show any differences in extracellular matrix deposition. sibilities by which VOCs may potentially activate immune Furthermore, total BAL cellularity and cytokine contents in BAL system pathways, our working hypothesis was that airway supernatant were not altered. In all, the data ruled out any detecta- hyperresponsiveness was underlain by an inflammatory response ble airway disease in the form of inflammation, increased mucus as generally is the case in airway disease. The inflammatory production, or remodeling. We also did not find signs of inflamma- silence revealed by our data in all lung compartments was, there- tion at other locations in the lung tissue sections, such as perivascu- fore, a puzzling outcome to us. In the human investigations, 8- lar or in the parenchyma. VOCs cannot act as antigens due to their isoprostane, VEGF, and bFGF were increased in exhaled breath low molecular weight but may still initiate an adaptive immune condensate, but no evidence of inflammatory activity was found Environmental Health Perspectives 027003-10 128(2) February 2020 Figure 10. Vascular endothelial growth factor (VEGF) immunostaining and quantitative morphology. (A) VEGF immunostaining (red signal) in the experi- mental Control, Exposed, and Rested groups of the Wistar and Brown Norway rats, as indicated. Counterstain: hematoxylin QS. The scale bar (50 lm) in the upper-left panel applies to all micrographs. (B) Numerical density of VEGF+ cells in the alveolar walls of the respective experimental groups and rat strains, as indicated. *p < 0:05 vs. Control. One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test for pairwise post-ANOVA comparisons; n = 6 for all experimental groups. †p < 0:05 vs. Wistar for independent interstrain group comparison (Student’s t-test). as per cytokine analysis (Rodríguez-Trigo et al. 2010). It is note- stimulus (Cheung et al. 1997). This mechanism has been proposed worthy that our study design did not include any group aimed at to contribute to airway narrowing in chronic obstructive pulmo- analyzing acute respiratory tract inflammation at an early- nary disease (COPD) (Koyama et al. 1996; Yang and Lin 2010), exposure time point. Therefore, our study cannot rule out the pos- although the usual centrilobular distribution of emphysema directly sibility of an early inflammatory response with subsequent down- affects the airway alveolar attachments in COPD, and airway inflam- regulation. Part of the previous work on the respiratory system mation is often present (Verhoeven et al. 2000). To verify the pres- effects of VOCs inhalation from oil spills focused on acute effects ence of a biological mechanism driving pulmonary emphysema, we (Alexander et al. 2018; Campbell et al. 1993; Janjua et al. 2006; performed in situ detection of apoptosis by the TUNEL technique on Lyons et al. 1999; Morita et al. 1999), yet there is a significant the tissue sections. Quantitative morphology revealed a baseline fre- knowledge gap on the inflammatory response associated with such quency of apoptotic alveolar septal cells in the control animals, at- outcomes. Such studies have reported symptom descriptions, with tributable to physiological cell turnover and consistent with previous few collecting spirometric data, blood hematology and biochemis- reports on nonsmoker subjects (Kasahara et al. 2001; Majo et al. try, or exposure analytes in urine, and did not attempt to gain 2001) and nonexposed animals in rat models of cigarette smoke- insights on the biological mechanisms of the acute respiratory induced emphysema (Kasahara et al. 2000; Li et al. 2017). We found response through assessments of respiratory tract inflammation. significantly increased numbers of apoptotic alveolar septal cells in Similarly, data on persistent biological mechanisms at a late stage the exposed animals of both rat strains, which persisted after the after exposure, such as in the time frame of the studies that followed washout. Overall, the data suggest that the inhaled fuel oil–derived the Prestige tank wreckage, are very limited (Rodríguez-Trigo et al. VOCs mainly interacted with the lung tissues at the most distal level, 2010). regionally affecting the peripheral parenchyma through mechanisms We unexpectedly found pulmonary emphysema, with a partic- that may not involve inflammation. In several animal models of ular and unusual pattern of subpleural distribution, developed by COPD, emphysema developed despite a noticeable absence of pul- both the Wistar and BN Exposed groups. Such emphysema was monary inflammation, suggesting that apoptosis of alveolar wall present at the end-of-exposure cutoff, and the quantitative data sug- cells is sufficient to cause emphysema without the formation of gest further progression by the end of the washout period in the inflammatory infiltrates (Demedts et al. 2006). CD8+ T cells have Rested groups. On a per-individual assessment basis, all exposed been reported to be increased in the lung parenchyma of COPD sub- rats were affected. This finding suggests an explanation for airway jects, not forming apparent inflammatory infiltrates, and CD8+ T- hyperresponsiveness in the absence of concomitant inflammation cell-mediated cytotoxicity has been proposed as a mechanism induc- on the basis of known pulmonary mechanics. The destruction of al- ing apoptosis of alveolar wall cells (Majo et al. 2001; Saetta et al. veolar walls decreases the elastic recoil of the lung parenchyma 1999). Contrary to this proposed mechanism, our CD8+ T-cell im- and facilitates airway narrowing upon a bronchoconstrictive munostaining and quantification yielded inconclusive data in the Environmental Health Perspectives 027003-11 128(2) February 2020 Exposed groups and showed a marked decrease in the Rested groups damage and development of emphysema in the absence of inflam- for both rat strains. It is unclear whether the CD8+ T-cell decrease in mation. However, there is no obvious interpretation of the flat per- the Rested groups may be part of a homeostatic mechanism to inhibit sistence of reduced VEFG expression in the Rested groups, overall cell-mediated cytotoxicity. Previous studies on humans particularly considering the septal endothelium recovery seen in the (Tanabe et al. 2012; Mohamed Hoesein et al. 2013) and COPD ani- Wistar rats, which suggests the involvement of alternative, undeter- mal models (Wright et al. 1994; March et al. 2006) demonstrated the mined pathways with significant capacities for the maintenance of persistence or progression of emphysema after cessation of exposure the alveolar structures. The Rested groups’ data also mismatch the to cigarette smoke but did not attempt to analyze the biological VEGF increases reported in exhaled breath condensate from the response of the parenchyma to injury interruption once emphysema Prestige VOCs-exposed subjects (Rodríguez-Trigo et al. 2010). was established. An alternative mechanism inducing alveolar septal In summary, experimental inhalational exposure of rats to VOCs cell apoptosis in our fuel oil VOCs exposure model may be DNA released by a fuel oil mimicking the Prestige tanker spill induced air- damage, which was detected in blood cells of subjects involved in way hyperresponsiveness, alveolar septal cell apoptosis, and pulmo- the handling of oil-contaminated birds from the Prestige spill nary emphysema with parenchymal microvascular endothelial (Laffon et al. 2006). DNA damage has been pointed out as a mecha- damage in the absence of inflammation and any signs of airway dis- nism inducing apoptosis of alveolar structural cells, leading to pul- ease. All effects were present at the end of a 3-wk exposure period monary emphysema (Demedts et al. 2006; Morissette et al. 2009). and persisted with emphysema progression after a 2-wk washout, Airflow dynamics during breathing may have favored higher and suggesting a self-sustained disease mechanism. The presence of air- more lasting VOC concentrations in the most peripheral regions of way hyperresponsiveness after the washout, with undetectable signs the lung parenchyma, where a critical threshold of DNA damage of concomitant inflammatory activity, suggests an analogy with the may have been surpassed. One open question regards the mecha- data from the studies conducted on human subjects in the affected nisms sustaining alveolar septal cell apoptotic activity and the pro- areas with a 2-y lapse after the catastrophe. The pulmonary emphy- gression of emphysema after the exposure washout. The finding of sema, with an unusual pattern of distribution over the most periph- increased levels of 8-isoprostane in exhaled breath condensate of the eral areas of the lung parenchyma, suggests a lung mechanics exposed subjects 2 y after the exposure (Rodríguez-Trigo et al. explanation for the noninflammatory airway hyperresponsiveness. 2010) is most probably an indicator of persistent oxidative stress in Although with limited evidence, the studies on humans, combined the pulmonary milieu. Pulmonary emphysema has been proposed to with the outcomes from our experimental model, allow us to specu- be proinflammatory by itself (Tokairin et al. 2008), which suggests late that DNA damage and oxidative stress may have been the driv- the hypothesis that, following its induction by VOCs exposure, the ing mechanisms leading to alveolar septal cell apoptosis and the persistence of cumulative DNA damage is aided by further tissue development of a pulmonary emphysema pattern different from stress factors to maintain increased alveolar septal cell apoptosis as a typical cigarette smoke–induced emphysema in its distribution self-sustaining condition. and pathophysiology. Our experimental model, in line with the The involvement of apoptosis in the pathogenesis of pulmonary available human data, supports the idea that inhalational exposure emphysema affects both principal types of structural alveolar septal to VOCs from fuel oil spill catastrophes may elicit persistent re- cells, i.e., type I pneumocytes and endothelial cells (Imai et al. 2005; spiratory disease and provides a plausible postulate for the patho- Kasahara et al. 2001; Segura-Valdez et al. 2000). Endothelial dam- physiological mechanisms involved. The data warrant effective age through apoptosis may indeed be an important driving force in respiratory protection measures and provide suggested guidance the development of emphysema (Demedts et al. 2006). For this rea- for clinical investigations and follow-up upon potential spill inci- son, we analyzed the volume density of CD31-immunostained en- dents in the future. dothelium in the alveolar walls. Consistent with the peripheral emphysema pattern in our model, the endothelium volume density Acknowledgments was severely reduced in the subpleural parenchymal regions. The This work was funded by the Xunta de Galicia (grants quantitative data also revealed, to a lesser degree, a significant 07CSA048916PR and INCITE07PXI916208E), Sociedad Española decrease of endothelium density in the otherwise apparently unaf- de Neumología y Cirugía Torácica (SEPAR) (grant 05/107), and fected central parenchyma. Remarkably, the Wistar Rested group Instituto de Salud Carlos III (grants PI08/1822, FI07/00399, and showed significant re-endothelization of the remnant alveolar walls FI05/00171) of Spain. The work was surveyed by the Instituto in the severely affected subpleural regions. This finding does not Tecnológico de Galicia (ITG) as an independent observer and plausibly suggest an emphysema repair mechanism but may reflect promoter entity (EPO). We thank the Repsol Technology Center a regenerative response aimed at some functional gas exchange re- in Madrid (Spain) for kindly synthesizing the fuel oil employed; covery of the alveolar wall remnants after the emphysema-inducing Electro-Medical Measurement Systems (EMMS) engineers, insult has ceased. The strain difference preventing the BN rat from especially Peter Connor, for the efforts in researching and developing such endothelium recovery is unclear. To our knowl- manufacturing the experimental exposure equipment; and the edge, the possibility of postinjury microvascular regeneration in Central Research Support Services of Universidade da Coruña emphysematous lungs has not been hypothesized, whether in ex- for their guidance and gas spectrometry analysis. smoker COPD subjects or animal models. We observed strong inhi- bition of VEGF expression in the Exposed and Rested groups of both strains. 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