Lung function of primary cooks using LPG or biomass and the effect of particulate matter on airway epithelial barrier integrity

Abstract

Background: Cooks exposed to biomass fuel experience increased risk of respiratory disease and mortality. We sought to characterize lung function and environmental exposures of primary cooking women using two fuel-types in southeastern India, as well as to investigate the effect of particulate matter (PM) from kitchens on human airway epithelial (HAE) cells in vitro.

Methods: We assessed pre- and post-bronchodilator lung function on 25 primary female cooks using wood biomass or liquified petroleum gas (LPG), and quantified exposures from 34 kitchens (PM_2.5, PM < 40 μm, black carbon, endotoxin, and PM metal and bacterial content). We then challenged HAE cells with PM, assessing its cytotoxicity to small-airway cells (A549) and its effect on: transepithelial conductance and macromolecule permeability (NuLi cells), and antimicrobial activity (using airway surface liquid, ASL, from primary HAE cells).

Results: Lung function was impaired in cooks using both fuel-types. 60% of participants in both fuel-types had respiratory restriction (post bronchodilator FEV₁/FVC>90). The remaining 40% in the LPG group had normal spirometry (post FEV₁/FVC = 80-90), while only 10% of participants in the biomass group had normal spirometry, and the remaining biomass cooks (30%) had respiratory obstruction (post FEV₁/FVC<80). Significant differences were found in environmental parameters, with biomass kitchens containing greater PM_2.5, black carbon, zirconium, arsenic, iron, vanadium, and endotoxin concentrations. LPG kitchens tended to have more bacteria (p = 0.14), and LPG kitchen PM had greater sulphur concentrations (p = 0.02). In vitro, PM induced cytotoxicity in HAE A549 cells in a dose-dependent manner, however the effect was minimal and there were no differences between fuel-types. PM from homes of participants with a restrictive physiology increased electrical conductance of NuLi HAE cells (p = 0.06) and decreased macromolar permeability (p ≤ 0.05), while PM from homes of those with respiratory obstruction tended to increase electrical conductance (p = 0.20) and permeability (p = 0.07). PM from homes of participants with normal spirometry did not affect conductance or permeability. PM from all homes tended to inhibit antimicrobial activity of primary HAE cell airway surface liquid (p = 0.06).

Conclusions: Biomass cooks had airway obstruction, and significantly greater concentrations of kitchen environmental contaminants than LPG kitchens. PM from homes of participants with respiratory restriction and obstruction altered airway cell barrier function, elucidating mechanisms potentially responsible for respiratory phenotypes observed in biomass cooks.

Keywords: Airway epithelial cell barrier integrity; Biomass; India; Particulate matter; Respiratory disease.

Figure 1.. Measurement design.

A. PM_2.5 measurements were conducted at approximate cooking height near the cookstove, arrow indicates device colocation B. EDCs collected endotoxin on a music stand (top arrow), while PM settled on pre-cleaned rugs below the EDC (bottom arrow) for 7 days C. Rugs were vacuumed into a sieve (arrow), collecting particles <40 μm D. One representative PM_2.5 daily time series from biomass home in log scale – peaks occurred during evening and morning hours, corresponding with typical times of evening meals, breakfast, and tea. Peak exposure exceeded 50 mg/m³, indicating extremely high peak concentrations.

Figure 2.. Post bronchodilator FEV₁/FVC by fuel-type.

Post FEV₁/FVC ratios from each participant, based on fuel-type (LPG n=5, Biomass n=20). FEV₁/FVC of 80 and 90 are represented by solid lines, between which lung function is considered normal, and circled values represent the participants with restrictive (>90), and obstructive (<80) lung function. The Mann-Whitney U test was used to determine differences in post bronchodilator FEV₁/FVC values between fuel-type.

Figure 3.. Total colony forming units by home.

Blank-corrected culturable bacterial CFUs, each dot represents total CFUs/mL per home found in PM (dose=1 mg/mL), by fuel-type.

Figure 4.. Cytotoxicity of a human airway epithelial cell-line exposed to LPG or Biomass kitchen PM.

Seven doses (4.7, 9.4, 18.8, 37.5, 75, 150, 300 μg/cm²) of PM from homes where spirometry and PM (n=22) was obtained were applied to A549 cells overnight, and percent cytotoxicity measured the following day.

Figure 5.. Conductance of human airway epithelial cells at air-liquid interface after exposure to PM from participants’ homes based on spirometry phenotype.

Change in 24-hr conductance (mS*cm²) after the NuLi HAE cell-line at ALI were exposed to PM from 22 homes. “Normal” indicates cells treated with PM from homes of participants with normal spirometry (post bronchodilator FEV₁/FVC = 80–90), “Rest.” indicates cells treated with PM from homes of participants with restrictive spirometry (post FEV₁/FVC >90) and “Obs.” indicates cells treated with homes of those with obstructive spirometry (post FEV₁/FVC <80). Results from cells treated with PM from homes of participants with normal spirometry were compared to results from cells treated with PM from obstructive and restrictive homes using Mann-Whitney U tests, and the dotted line indicates conductance of control (vehicle treated) cells.

Figure 6.. FITC dextran permeability of human airway epithelial cells at air-liquid interface after exposure to PM from participants’ homes based on spirometry phenotype.

Permeability of FITC-D after 24 hours, collected from basolateral media of NuLi HAE cell-line at ALI after exposure to 30 μg/cm² PM, grouped by participants’ spirometry results. “Normal” indicates cells treated with PM from homes of participants with normal spirometry (post bronchodilator FEV₁/FVC = 80–90), “Rest.” indicates cells treated with PM from homes of participants with restrictive spirometry (post FEV₁/FVC >90) and “Obs.” indicates cells treated with homes of those with obstructive spirometry (post FEV₁/FVC <80). The dotted line indicates control permeability. Cells treated with PM from “normal” spirometry results were compared to PM from restrictive homes using a Mann-Whitney U test and compared to PM from obstructive homes using an unpaired t-test.

Figure 7.. Antimicrobial activity of airway surface liquid with and without exposure to PM from cooks’ homes.

Percent of live S. aureus exposed to three human donor’s ASL + vehicle after 10 minutes compared to the percent of live bacteria after ASL is exposed to PM from cooks’ homes (each dot represents summary data from 3 donor ASL for each of 19 homes), Mann-Whitney U test comparing ASL alone (n=3) to all PM-treated ASL (n=19), p=0.06.