Environmentally persistent free radicals: Methods for combustion generation, whole-body inhalation and assessing cardiopulmonary consequences.

Particulate matter (PM) containing environmentally persistent free radicals (EPFRs) results from the incomplete combustion of organic wastes which chemisorb to transition metals. This process generates a particle-pollutant complex that continuously redox cycles to produce reactive oxygen species. EPFRs are well characterized, but their cardiopulmonary effects remain unknown. This publication provides a detailed approach to evaluating these effects and demonstrates the impact that EPFRs have on the lungs and vasculature. Combustion-derived EPFRs were generated (EPFR lo: 2.1e radical/g, EPFR hi: 5.5e radical/g), characterized, and verified as representative of those found in urban areas. Dry particle aerosolization and whole-body inhalation were established for rodent exposures. To verify that these particles and exposures recapitulate findings relevant to known PM-induced cardiopulmonary effects, male C57BL6 mice were exposed to filtered air, ∼280 μg/m EPFR lo or EPFR hi for 4 h/d for 5 consecutive days. Compared to filtered air, pulmonary resistance was increased in mice exposed to EPFR hi. Mice exposed to EPFR hi also exhibited increased plasma endothelin-1 (44.6 vs 30.6 pg/mL) and reduced nitric oxide (137 nM vs 236 nM), suggesting vascular dysfunction. Assessment of vascular response demonstrated an impairment in endothelium-dependent vasorelaxation, with maximum relaxation decreased from 80% to 62% in filtered air vs EPFR hi exposed mice. Gene expression analysis highlighted fold changes in aryl hydrocarbon receptor (AhR) and antioxidant response genes including increases in lung Cyp1a1 (8.7 fold), Cyp1b1 (9 fold), Aldh3a1 (1.7 fold) and Nqo1 (2.4 fold) and Gclc (1.3 fold), and in aortic Cyp1a1 (5.3 fold) in mice exposed to EPFR hi vs filtered air. We then determined that lung AT2 cells were the predominate locus for AhR activation. Together, these data suggest the lung and vasculature as particular targets for the health impacts of EPFRs and demonstrate the importance of additional studies investigating the cardiopulmonary effects of EPFRs.