Differential expression of pro-inflammatory and oxidative stress mediators induced by nitrogen dioxide and ozone in primary human bronchial epithelial cells

Abstract

Context: NO2 and O3 are ubiquitous air toxicants capable of inducing lung damage to the respiratory epithelium. Due to their oxidizing capabilities, these pollutants have been proposed to target specific biological pathways, but few publications have compared the pathways activated.

Objective: This work will test the premise that NO2 and O3 induce toxicity by activating similar cellular pathways.

Methods: Primary human bronchial epithelial cells (HBECs, n = 3 donors) were exposed for 2 h at an air-liquid interface to 3 ppm NO2, 0.75 ppm O3, or filtered air and harvested 1 h post-exposure. To give an overview of pathways that may be influenced by each exposure, gene expression was measured using PCR arrays for toxicity and oxidative stress. Based on the results, genes were selected to quantify whether expression changes were changed in a dose- and time-response manner using NO2 (1, 2, 3, or 5 ppm), O3 (0.25, 0.50, 0.75, or 1.00 ppm), or filtered air and harvesting 0, 1, 4 and 24 h post-exposure.

Results: Using the arrays, genes related to oxidative stress were highly induced with NO2 while expression of pro-inflammatory and vascular function genes was found subsequent to O3. NO2 elicited the greatest HMOX1 response, whereas O3 more greatly induced IL-6, IL-8 and PTGS2 expression. Additionally, O3 elicited a greater response 1 h post-exposure and NO2 produced a maximal response after 4 h.

Conclusion: We have demonstrated that these two oxidant gases stimulate differing mechanistic responses in vitro and these responses occur at dissimilar times.

Keywords: In vitro; inflammation; nitrogen dioxide; oxidant; oxidative stress; ozone.

Figure 1. Gene expression heat maps generated using a PrimePCR Pathway Plate of the Stress and Toxicity SAB Target List (Bio-Rad)

A total of 84 genes were assessed following exposures to 0.75 ppm O₃ (n = 3) or 3 ppm NO₂ (n = 3). Using a cut-off of a 1.5 fold increase or decrease, 33 genes were differentially upregulated or downregulated between the 2 exposure scenarios. Fold changes were calculated relative to a filtered air control.

Figure 2. Gene expression heat maps generated using a Human Oxidative Stress and Antioxidant Defense RT² Profiler PCR Array (SABiosciences)

A total of 84 genes were assessed following exposures to 0.75 ppm O₃ (n = 3) or 3 ppm NO₂ (n = 3). Using a cut-off of a 1.5 fold increase or decrease, 27 genes were differentially upregulated or downregulated between the 2 exposure scenarios. Fold changes were calculated relative to a filtered air control.

Figure 3. mRNA expression of IL-8 following 2 hr long exposures of HBECs to various concentrations of O₃ and NO₂ at A) 1 hr post exposure and b) 4 hrs post exposure

The data is expressed as the mRNA fold change relative to a filtered air control. Data were analyzed using 1-factor ANOVA for dose, followed by Tukey’s Multiple Comparison Post Test. p-values were calculated between treatments and the filtered air control. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001. N = 5 donors.

Figure 4. mRNA expression of PTGS2 following 2 hr long exposures of HBECs to various concentrations of O₃ and NO₂ at A) 1 hr post exposure and b) 4 hrs post exposure

The data is expressed as the mRNA fold change relative to a filtered air control. Data were analyzed using 1-factor ANOVA for dose, followed by Tukey’s Multiple Comparison Post Test. p-values were calculated between treatments and the filtered air control. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001. N = 5 donors.

Figure 5. mRNA expression of IL-6 following 2 hr long exposures of HBECs to various concentrations of O₃ and NO₂ at A) 1 hr post exposure and b) 4 hrs post exposure

The data is expressed as the mRNA fold change relative to a filtered air control. Data were analyzed using 1-factor ANOVA for dose. N = 3 donors.

Figure 6. mRNA expression of HMOX1 following 2 hr long exposures of HBECs to various concentrations of O₃ and NO₂ at A) 1 hr post exposure and b) 4 hrs post exposure

The data is expressed as the mRNA fold change relative to a filtered air control. Data were analyzed using 1-factor ANOVA for dose, followed by Tukey’s Multiple Comparison Post Test. p-values were calculated between treatments and the filtered air control. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001. N = 5 donors.