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

Abstract

Background: Fuel oil-derived volatile organic compounds (VOCs) inhalation is associated with accidental marine spills. After the Prestige petroleum tanker sank off northern Spain in 2002 and the Deepwater Horizon oil rig catastrophe in 2009, subjects involved in environmental decontamination 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 bronchoalveolar 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 morphology 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 factor (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.

Figure 1.

Airway responsiveness to methacholine challenge. The plots represent the pulmonary resistance ($R_L$) peak after each methacholine (MCh) dose. The Exposed animals inhaled fuel oil–derived VOCs for 2-h sessions, 5 times per week, for 3 wk. The Rested animals were subjected to the same VOC exposure regime followed by a 2-wk washout period. The Control (CTRL) animals had no exposure. These group definitions apply uniformly to the Wistar and Brown Norway rats and the C57BL/6 mice for all subsequent figures and data tables as applicable. $R_L$ values of (A) Wistar rats, (B) Brown Norway 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. Statistical analysis: one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test for pairwise post-ANOVA comparisons at each MCh concentration level; $n=6$ for all experimental groups.

Figure 2.

Bronchoalveolar lavage (BAL) analysis. Total BAL leukocytes were counted at the end of VOCs exposure (Exposed group) and after a 2-wk washout (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 $8.5\text{\hspace{0.17em} pg}/\mathrm{mL}$ for interleukin 1 alpha ($\text{IL-}1\mathrm{\alpha }$), $0.5\text{\hspace{0.17em} pg}/\mathrm{mL}$ for macrophage chemotactic protein 1 (MCP-1), $4.3\text{\hspace{0.17em} pg}/\mathrm{mL}$ for tumor necrosis $\text{factor-}\mathrm{\alpha }$ ($\text{TNF}\mathrm{\alpha }$), $8.3\text{\hspace{0.17em} pg}/\mathrm{mL}$ for interferon gamma ($\text{IFN-}\mathrm{\gamma }$), $5.0\text{\hspace{0.17em} pg}/\mathrm{mL}$ for granulocyte–monocyte colony stimulation factor (GM-CSF), and $0.3\text{\hspace{0.17em} pg}/\mathrm{mL}$ for IL-4. Graph bars represent 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 pairwise post-ANOVA comparisons; $n=6$ for all experimental groups.

Figure 3.

Airway histopathology in hematoxylin and eosin–stained tissue sections. Micrographs show representative cross-sectioned airways from the respective strains and experimental groups as indicated. Scale bars: $200\mathrm{\mu }\mathrm{m}$.

Figure 4.

Periodic acid–Schiff (PAS) and Masson’s trichrome staining. Lung tissue sections were stained with PAS to identify goblet cells and evaluate the overall airway epithelial mucus load. Masson’s trichrome was employed to evaluate extracellular matrix deposition. The panels show representative cross-sectioned airways for the respective stainings, animal strains, and experimental groups as indicated. No pathological alterations were identified in terms of goblet cell hyperplasia or hypertrophy, overall epithelial mucus load, or subepithelial fibrosis as a result of volatile organic compounds (VOCs) inhalation. Scale bar: $100\mathrm{\mu }\mathrm{m}$.

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 respective 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: $200\mathrm{\mu }\mathrm{m}$. *$p<0.05$ 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 pairwise post-ANOVA comparisons; $n=6$ for all experimental groups.

Figure 6.

Detection of apoptotic alveolar septal cells in rat lung parenchyma. Apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique on lung tissue sections. (A) Detail of an apoptotic alveolar septal cell, likely a type I pneumocyte, identified by its purple-stained nucleus with condensed chromatin. The bar graphs show the quantitative morphology data on the frequency of alveolar septal ${\text{TUNEL}}^{+}$ cells for the Control, Exposed, and Rested experimental groups in (B) the Wistar rat, and (C) the Brown Norway rat. Scale bar: $10\mathrm{\mu }\mathrm{m}$. *$p<0.05$ vs. Control (CTRL) group. 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.

Figure 7.

Distribution of apoptotic alveolar septal cells. For quantitative morphology, the terminal deoxynucleotidyl transferase dUTP nick end labeling (${\text{TUNEL}}^{+}$) alveolar septal cells were identified through high-resolution field sampling. The images shown here are low-magnification micrographs to illustrate 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: $50\mathrm{\mu }\mathrm{m}$.

Figure 8.

Quantification of ${\text{CD}8}^{+}$ T cells in lung parenchyma. $\text{CD}{8}^{+}$ cells were immunostained (red signal) and their numerical density quantified in the alveolar 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 $\text{CD}{8}^{+}$ cells can be identified. The image, from another control Wistar rat, is representative to provide a visual sense of the frequency of $\text{CD}{8}^{+}$ 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 contain any $\text{CD}{8}^{+}$ cells. The image shown here has intentionally captured one $\text{CD}{8}^{+}$ cell as a reference, visible in the upper-left quadrant. (D) Numerical density of $\text{CD}{8}^{+}$ T cells in the alveolar walls. *$p<0.05$ vs. Control (CTRL). ^†$p<0.05$ vs. Exposed. Scale bar: $50\mathrm{\mu }\mathrm{m}$ in (A); $100\mathrm{\mu }\mathrm{m}$ 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).

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 (subpleural) 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 ($50\mathrm{\mu }\mathrm{m}$) 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).

Figure 10.

Vascular endothelial growth factor (VEGF) immunostaining and quantitative morphology. (A) VEGF immunostaining (red signal) in the experimental Control, Exposed, and Rested groups of the Wistar and Brown Norway rats, as indicated. Counterstain: hematoxylin QS. The scale bar ($50\mathrm{\mu }\mathrm{m}$) in the upper-left panel applies to all micrographs. (B) Numerical density of ${\text{VEGF}}^{+}$ 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).