DataSheet1_Assessing susceptibility for polycyclic aromatic hydrocarbon toxicity in an in vitro 3D respiratory model for asthma.PDF

There is increased emphasis on understanding cumulative risk from the combined effects of chemical and non-chemical stressors as it relates to public health. Recent animal studies have identified pulmonary inflammation as a possible modifier and risk factor for chemical toxicity in the lung after exposure to inhaled pollutants; however, little is known about specific interactions and potential mechanisms of action. In this study, primary human bronchial epithelial cells (HBEC) cultured in 3D at the air-liquid interface (ALI) are utilized as a physiologically relevant model to evaluate the effects of inflammation on toxicity of polycyclic aromatic hydrocarbons (PAHs), a class of contaminants generated from incomplete combustion of fossil fuels. Normal HBEC were differentiated in the presence of IL-13 for 14 days to induce a profibrotic phenotype similar to asthma. Fully differentiated normal and IL-13 phenotype HBEC were treated with benzo[a]pyrene (BAP; 1–40 μg/mL) or 1% DMSO/PBS vehicle at the ALI for 48 h. Cells were evaluated for cytotoxicity, barrier integrity, and transcriptional biomarkers of chemical metabolism and inflammation by quantitative PCR. Cells with the IL-13 phenotype treated with BAP result in significantly (p < 0.05) decreased barrier integrity, less than 50% compared to normal cells. The effect of BAP in the IL-13 phenotype was more apparent when evaluating transcriptional biomarkers of barrier integrity in addition to markers of mucus production, goblet cell hyperplasia, type 2 asthmatic inflammation and chemical metabolism, which all resulted in dose-dependent changes (p < 0.05) in the presence of BAP. Additionally, RNA sequencing data showed that the HBEC with the IL-13 phenotype may have increased potential for uncontrolled proliferation and decreased capacity for immune response after BAP exposure compared to normal phenotype HBEC. These data are the first to evaluate the role of combined environmental factors associated with inflammation from pre-existing disease and PAH exposure on pulmonary toxicity in a physiologically relevant human in vitro model.</p

Time line for the experimental protocol.

<p>Mice continued to eat their respective foods throughout the allergen sensitization and challenge period. They were sensitized with saline or OVA at 8 and 10 weeks, and then challenged by nebulization with saline or OVA once at 10 weeks and on three consecutive days during week 12. Mice were sacrificed 24 h after the final nebulization.</p

BAL fluid cytology proportions for mice fed (n-3) or (n-6) PUFA enriched foods for 8 weeks and then sensitized and challenged with OVA for an additional 4 weeks compared with mice fed control food that were sensitized and challenged with saline (Negative Control) or OVA (Positive Control)¹.

<p>BAL fluid cytology proportions for mice fed (n-3) or (n-6) PUFA enriched foods for 8 weeks and then sensitized and challenged with OVA for an additional 4 weeks compared with mice fed control food that were sensitized and challenged with saline (Negative Control) or OVA (Positive Control)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163819#t004fn001" target="_blank">¹</a>.</p

Composition of selected FA in diets fed to mice for 12 weeks.

<p>Composition of selected FA in diets fed to mice for 12 weeks.</p

Percent of lung tissue staining positive with periodic acid-Schiff (PAS) stain for mucus and rat anti-mouse CD45 stain, after mice were fed (n-3) or (n-6) PUFA enriched foods for 8 weeks and then sensitized and challenged with OVA for an additional 4 weeks compared with mice fed control food that were sensitized and challenged with saline (Negative Control) or OVA (Positive Control)¹.

<p>Percent of lung tissue staining positive with periodic acid-Schiff (PAS) stain for mucus and rat anti-mouse CD45 stain, after mice were fed (n-3) or (n-6) PUFA enriched foods for 8 weeks and then sensitized and challenged with OVA for an additional 4 weeks compared with mice fed control food that were sensitized and challenged with saline (Negative Control) or OVA (Positive Control)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163819#t006fn001" target="_blank">¹</a>.</p

Representative light microscopy images of lung tissue.

<p>Lung tissues were stained with rat anti-mouse CD45 after mice were fed their respective foods for 12 weeks. They were sensitized and challenged with saline or OVA starting at 8 weeks. (A) Only circulating leukocytes are seen in control mice fed standard rodent food and sensitized and challenged with saline (Negative Control). (B) Mice fed standard rodent food and sensitized and challenged with OVA (Positive Control) or (C) Corn Oil-enriched food exhibit a marked increase in CD45+ inflammatory cells, particularly peribronchial infiltrates. (D) Overall, there were less CD45+ inflammatory cells in mice fed Fish-Oil enriched food compared with mice fed control food (Positive Control; <i>P</i> = 0.04). Original magnification 25X.</p

Plasma concentrations (g/100 g FA) of selected FA in mice after dietary intervention for 12 weeks¹.

<p>Plasma concentrations (g/100 g FA) of selected FA in mice after dietary intervention for 12 weeks<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163819#t003fn001" target="_blank">¹</a>.</p

Cytokine (IL-4 and IL-13) concentrations in BAL fluid of mice fed (n-3) or (n-6) PUFA enriched foods for 8 weeks and then sensitized and challenged with OVA for an additional 4 weeks compared with mice fed control food that were sensitized and challenged with saline (Negative Control) or OVA (Positive Control)¹.

<p>Cytokine (IL-4 and IL-13) concentrations in BAL fluid of mice fed (n-3) or (n-6) PUFA enriched foods for 8 weeks and then sensitized and challenged with OVA for an additional 4 weeks compared with mice fed control food that were sensitized and challenged with saline (Negative Control) or OVA (Positive Control)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163819#t005fn001" target="_blank">¹</a>.</p

Average nutrient composition (dry matter basis) of diets fed to mice for 12 weeks.

<p>Average nutrient composition (dry matter basis) of diets fed to mice for 12 weeks.</p