Aerosol physicochemical determinants of carbon black and ozone inhalation co-exposure induced pulmonary toxicity

Abstract

Air pollution accounts for more than 7 million premature deaths worldwide. Using ultrafine carbon black (CB) and ozone (O3) as a model for an environmental co-exposure scenario, the dose response relationships in acute pulmonary injury and inflammation were determined by generating, characterizing, and comparing stable concentrations of CB aerosols (2.5, 5.0, 10.0 mg/m3), O3 (0.5, 1.0, 2.0 ppm) with mixture CB + O3 (2.5 + 0.5, 5.0 + 1.0, 10.0 + 2.0). C57BL6 male mice were exposed for 3 h by whole body inhalation and acute toxicity determined after 24 h. CB itself did not cause any alteration, however, a dose response in pulmonary injury/inflammation was observed with O3 and CB + O3. This increase in response with mixtures was not dependent on the uptake but was due to enhanced reactivity of the particles. Benchmark dose modeling showed several-fold increase in potency with CB + O3 compared with CB or O3 alone. Principal component analysis provided insight into response relationships between various doses and treatments. There was a significant correlation in lung responses with charge-based size distribution, total/alveolar deposition, oxidant generation, and antioxidant depletion potential. Lung tissue gene/protein response demonstrated distinct patterns that are better predicted by either particle dose/aerosol responses (interleukin-1β, keratinocyte chemoattractant, transforming growth factor beta) or particle reactivity (thymic stromal lymphopoietin, interleukin-13, interleukin-6). Hierarchical clustering showed a distinct signature with high dose and a similarity in mRNA expression pattern of low and medium doses of CB + O3. In conclusion, we demonstrate that the biological outcomes from CB + O3 co-exposure are significantly greater than individual exposures over a range of aerosol concentrations and aerosol characteristics can predict biological outcome.

Keywords: co-exposure; inflammation; inhalation; ozone; physicochemical properties; ultrafine carbon black.

Figure 1.

Aerosol exposure and characterization. A, Real-time monitoring of CB and O₃ levels during different exposures. B, Aerosol size distribution of CB (2.5, 5.0, and 10.0 mg/m³) and CB + O₃ (2.5 mg/m³ + 0.5 ppm, 5.0 mg/m³ + 1.0 ppm, 10.0 mg/m³ + 2.0 ppm) aerosol particles collected from the inhalation exposure chamber using an aerosol particle sizer (APS) in combination with scanning mobility particle sizer (SMPS) and an electrical low-pressure impactor (ELPI+).

Figure 2.

Acellular characterization of aerosol reactivity. A, XPS analysis representing surface oxygen contents of CB and CB + O₃ particles at low (2.5 mg/m³ + 0.5 ppm), medium (5 mg/m³ + 1 ppm) and high (10 mg/m³ + 2 ppm) dose. B, Representative X-band electron paramagnetic resonance (EPR) spectra of CM• in PBS with single (CB) and co-exposure (CB + O₃) particles suspension (50 μg/ml) at low (2.5 mg/m³ + 0.5 ppm), medium (5 mg/m³ + 1 ppm), and high (10 mg/m³ + 2 ppm) dose. Histogram represents intensity of the first peak of EPR signals. C) FRAS assay of single (CB) and co-exposure (CB + O₃) aerosol at low (2.5 mg/m³ + 0.5 ppm), medium (5 mg/m³ + 1 ppm), and high (10 mg/m³ + 2 ppm) exposure dose. Data are presented as mean ± SEM of 3 independent experiments. Data analyzed by 1-way ANOVA followed by Tukey’s post hoc test. *p < .05.

Figure 3.

Histopathological assessments of lung tissue after low and medium dose exposures. Lung tissues were collected 24 h post-exposure, fixed in neutral buffered formalin, and stained with H&E. Light photomicrographs of sections from (A) filtered air, (B) CB 2.5 mg/m³, (C) O₃ 0.5 ppm, (D) CB + O₃ (2.5 mg/m³ + 0.5 ppm), (E) CB 5.0 mg/m³, (F) O₃ 1.0 ppm, (G) CB +O₃ (5.0 mg/m³ + 1.0 ppm). Tissues were semi-quantitatively scored in a blinded manner by a board-certified veterinary pathologist for (H) inflammatory cell influx, (I) airway epithelial injury/cell death, and (J) macrophage hyperplasia. b, bronchiole; tb, terminal bronchiole; ad, alveolar duct; v, blood vessel; stippled arrows, inflammatory cell infiltration; solid arrows, necrotic/exfoliated airway epithelial cells. Insets on (D), (F), and (G) are higher magnification on the areas where pathological lesions are noted.

Figure 4.

Score plot of the first 2 components of the principal component analysis showing pulmonary injury and inflammatory response after exposure to air (control), CB, O₃, and CB + O₃ at low (2.5 mg/m³ and/or 0.5 ppm), medium (5 mg/m³ and/or 1 ppm), and high (10 mg/m³ and/or 2 ppm), for 3 h followed by euthanasia 24 h post-exposure. The response of animals from each group and their clustering was highlighted qualitatively by the oval.

Figure 5.

Particle uptake. A, Representative images of in vivo uptake of particles (CB and CB + O₃) by broncho-alveolar lavage macrophages (×40 and ×100 magnification) at low (0.5 ppm and/or 2.5 mg/m³), medium (1 ppm and/or 5 mg/m³), and high (2 ppm and/or 10 mg/m³) exposures for 3 h, followed by euthanasia 24 h post-exposure. Quantification of particle uptake by (B) percentage particle positive macrophages and (C) percentage of particle area in macrophages. D, Lung burden quantification of particles (CB and CB + O₃) 24 h post-exposure. Data analyzed by 2-way ANOVA followed by Tukey’s post hoc test. *p < .05 versus CB + O₃ at same dose, ^p ≤ .05 low versus medium dose, ^$p ≤ .05 medium versus high dose, ^&p < .05 low versus high dose. <DL denotes lower than detection limit of the assay (0.8 μg).

Figure 6.

Multivariate analysis linking aerosol characteristics and oxidative properties of the mixtures to pulmonary injury and inflammation. A) Heat map representing Pearson correlations of aerosol characteristics and oxidative characteristics of the mixtures with biological outcomes. B) Table outlining the top correlated biological variables with aerosol characteristics and oxidative characteristics based on p-value.

Figure 7.

Lung tissue real-time mRNA expression. A) Heat Map representing the fold changes of mRNA gene expression in lung tissue after exposure to air (control), CB, O₃, and CB + O₃ at low (2.5 mg/m³ and/or 0.5 ppm), medium (5.0 mg/m³ and/or 1 ppm), and high (10.0 mg/m³ and/or 2.0 ppm), for 3 h, followed by euthanasia 24 h post-exposure. The values are normalized to Log2-fold change. Data analyzed by 2-way ANOVA followed by Tukey’s post hoc test. *p < .05 versus control, #p ≤ .05 versus CB + O₃ single exposure at same dose, ^p ≤ .05 CB + O₃ low versus high dose, $p ≤ .05 CB + O₃ medium versus high dose, &p ≤ .05 CB + O₃ low versus medium dose. B) Hierarchical clustering analysis (HCA) of mRNA gene expression after exposure to air (control) and CB + O₃ at low (2.5 mg/m³ + 0.5 ppm), medium (5.0 mg/m³ + 1.0 ppm), and high (10.0 mg/m³ + 2.0 ppm) doses, for 3 h, followed by euthanasia 24 h post-exposure.

Figure 8.

Predictor screening of broncho-alveolar lavage cytokines. A) Heat map representing the cytokine quantification in broncho-alveolar lavage fluid after exposure to air (control), CB, O₃, and CB + O₃ at low (2.5 mg/m³ and/or 0.5 ppm), medium (5.0 mg/m³ and/or 1.0 ppm), and high (10.0 mg/m³ and/or 2.0 ppm) doses for 3 h followed by euthanasia 24 h post-exposure. B) Heat map representing the cytokine quantification in lung tissue homogenate after exposure to air (control), CB, O₃, and CB + O₃ at low (2.5 mg/m³ and/or 0.5 ppm), medium (5.0 mg/m³ and/or 1.0 ppm), and high (10.0 mg/m³ and/or 2.0 ppm) doses for 3 h followed by euthanasia 24 h post-exposure The values are normalized to Log2 fold change. Data analyzed by 2-way ANOVA followed by Tukey’s post hoc test. *p < .05 versus control, #p ≤ .05 CB + O₃ versus single exposure at same dose, ^p ≤ .05 CB + O₃ low versus high dose, $p ≤ .05 CB + O₃ medium versus high dose, &p ≤ .05 CB + O₃ low versus medium dose. C) Heat map representing percentage contribution of aerosol characteristics in cytokine production based on boot strap forest approach.

Graphical abstract