Transcriptomics of single dose and repeated carbon black and ozone inhalation co-exposure highlight progressive pulmonary mitochondrial dysfunction

Abstract

Background Air pollution is a complex mixture of particles and gases, yet current regulations are based on single toxicant levels failing to consider potential interactive outcomes of co-exposures. We examined transcriptomic changes after inhalation co-exposure to a particulate and a gaseous component of air pollution and hypothesized that co-exposure would induce significantly greater impairments to mitochondrial bioenergetics. A whole-body inhalation exposure to ultrafine carbon black (CB), and ozone (O3) was performed, and the impact of single and multiple exposures was studied at relevant deposition levels. C57BL/6 mice were exposed to CB (10 mg/m3) and/or O3 (2 ppm) for 3 h (either a single exposure or four independent exposures). RNA was isolated from lungs and mRNA sequencing performed using the Illumina HiSeq. Lung pathology was evaluated by histology and immunohistochemistry. Electron transport chain (ETC) activities, electron flow, hydrogen peroxide production, and ATP content were assessed. Results Compared to individual exposure groups, co-exposure induced significantly greater neutrophils and protein levels in broncho-alveolar lavage fluid as well as a significant increase in mRNA expression of oxidative stress and inflammation related genes. Similarly, a significant increase in hydrogen peroxide production was observed after co-exposure. After single and four exposures, co-exposure revealed a greater number of differentially expressed genes (2251 and 4072, respectively). Of these genes, 1188 (single exposure) and 2061 (four exposures) were uniquely differentially expressed, with 35 mitochondrial ETC mRNA transcripts significantly impacted after four exposures. Both O3 and co-exposure treatment significantly reduced ETC maximal activity for complexes I (− 39.3% and − 36.2%, respectively) and IV (− 55.1% and − 57.1%, respectively). Only co-exposure reduced ATP Synthase activity (− 35.7%) and total ATP content (30%). Further, the ability for ATP Synthase to function is limited by reduced electron flow (− 25%) and translation of subunits, such as ATP5F1, following co-exposure. Conclusions CB and O3 co-exposure cause unique transcriptomic changes in the lungs that are characterized by functional deficits to mitochondrial bioenergetics. Alterations to ATP Synthase function and mitochondrial electron flow underly a pathological adaptation to lung injury induced by co-exposure.

Lung-gut axis of microbiome alterations following co-exposure to ultrafine carbon black and ozone

“…The particle size distribution was measured from the exposure chamber using the (1) Electrical Low-Pressure Impactor (ELPI+, Dakati, Tempera, Finland), (2) Aerosol Particle Sizer (APS 3321, TSI Inc Shoreview, MN), 3) Scanning mobility particle sizer (SMPS 3938, TSI Inc. Shoreview, MN), and 4) Nano Micro-orifice Uniform Deposit Impactor (Moudi 115R, MSP Corp, Shoreview, MN). The morphology of the aerosolized particles was observed using a field-emission scanning electron microscope (Hitachi S4800, Tokyo, Japan) and transmission electron microcopy (JEOL JEM-2100 TEM) as described previously [ 3 ].…”

“…The SpectraMax® iD5 (Molecular Devices, CA) plate reader was used to measure the absorbance after 45 min incubation, at 560 nm wavelength. The results are normalized to total protein concentration measured by BCA (bicinchoninic acid) assay kit as described previously [ 3 , 57 ].…”

“…The toxicity of the individual components of air pollution (PM, O 3 , NOx- Nitrogen oxides) are well established and form the basis of current regulatory decision making. However, interactive outcomes between different components of air pollution are not well established [ 3 ]. We previously reported that ozone (O 3 ) and carbon black (CB) inhalation co-exposure induces significantly greater lung injury and inflammation as well as macrophage driven endothelial cell damage [ 4 ].…”

“…We recently reported that repeated inhalation exposure to ultrafine CB and O 3 caused pulmonary injury and progressive mitochondrial dysfunction [ 3 ]. The present study was designed to define changes in the lung and gut microbiome after single or repeated inhalation exposure in male C57BL/6J mice.…”

Editorial: Exploring impacts of combined exposures to particles and chemicals on immune reactions across living organisms

“…Current regulations limit single toxicant levels but do not consider potential interactive effects of oxidant gases and nanomaterials, where both are known to develop their respiratory toxicity via the local generation of oxidative stress. While ozone is already well known to cause injury to the fragile alveolar region of the lungs, co-exposure can amplify single toxicant outcomes, as observed in a mouse study from Hathaway and colleagues, where progressive pulmonary mitochondrial dysfunction has been described ( Hathaway et al, 2021 ). Eventually, volatile organic compounds (VOCs) can be released during manufacturing processes and are present in several products (paints, solvents, glues, gasoline) ( Alijagic et al ).…”