Lipid-soluble components in cigarette smoke induce mitochondrial production of reactive oxygen species in lung epithelial cells

Abstract

Reactive oxygen species (ROS) present in cigarette smoke (CS) are thought to contribute to the development of COPD. Although CS-ROS can hardly enter airway epithelial cells, and certainly not the circulation, systemic levels of ROS have been found to be elevated in COPD patients. We hypothesize that lipophilic components present in CS can enter airway epithelial cells and increase intracellular ROS production by disturbing mitochondrial function. Different airway epithelial cells were exposed to CS extract (CSE), hexane-treated CSE (CSE without lipophilic components), gaseous-phase CS, and water-filtered CS (gaseous-phase CS without ROS). Mitochondrial membrane potential (Deltapsi(m)) and ATP levels were assessed using the bronchial epithelial cell line Beas-2b. ROS generation measured directly by DCF fluorescence and indirectly by measuring free thiol groups (-SH) upon exposure to CS was assessed using lung alveolar epithelial cells devoid of functional mitochondria (A549-rho0), with normal A549 cells serving as controls. In Beas-2b cells, CSE (4 h) caused a dose-dependent decrease in Deltapsi(m) and ATP levels, whereas hexane-treated CSE did not. DCF fluorescence in A549 cells increased in response to CSE, whereas this was not the case in A549-rho0 cells. Exposure of A549 cells to CS resulted in a rapid decrease in free -SH, whereas exposure to ROS-depleted CS only resulted in a delayed decrease. This delayed decrease was less pronounced in A549-rho0 cells. Lipophilic components in CS disturb mitochondrial function, which contributes to increased intracellular generation of ROS. Our results are of importance in understanding the systemic effects of smoking observed in patients with COPD.

Fig. 1.

Gaseous-phase cigarette smoke (CS) exposure system scheme. A: preparation of CS extract and the exposure of l-cysteine to air, CS, or water-filtered CS (WF-CS). B: exposure of airway epithelial cells to air, CS, and WF-CS.

Fig. 2.

Cigarette smoke extract (CSE) induces a loss of mitochondrial function in human bronchial epithelial cell line (Beas-2b). Effects of increasing concentrations of CSE and hexane-treated CSE (Hx-CSE) on mitochondrial membrane potential (Δψ_m; A) and intracellular ATP levels (B) in Beas-2b exposed for 4 h (n = 3) are shown. C: effects of increasing concentrations of lipophilic components on Δψ_m and intracellular ATP levels in Beas-2b exposed for 4 h (n = 4) are shown. P values given above the CSE lines represent the significance for the decline of Δψ_m and ATP levels. *P < 0.05 for comparison between the lipophilic components and DMSO control experiment. **P < 0.01 for comparison between the lipophilic components and DMSO control experiment.

Fig. 3.

CSE induces reactive oxygen species (ROS) generation in human alveolar epithelial cells (A549). Effects of CSE (20%) or PMA (10 ng/ml) on ROS generation in A549 and alveolar epithelial cells lacking a mitochondrial membrane potential (A549-ρO) are shown. Live cells were loaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCF) before being imaged on an inverted phase-contrast and fluorescence microscope. Representative images selected from randomly chosen fields are shown.

Fig. 4.

Effects of different oxidizing agents on the level of free thiol groups of l-cysteine in solution. A: incubation of 150 μM l-cysteine with increasing concentrations of H₂O₂ in the absence of light. B: control air (Air), CS, CS + vitamin C, and WF-CS are shown. Data (n = 4) are expressed as mean values ± SD. Free thiol groups were studied using Ellman's reagent. P values given above the lines in A and B represent the significance for the total decline of free thiol groups.

Fig. 5.

Effects of gaseous-phase CS and gaseous-phase WF-CS on free thiol groups in human alveolar epithelial cells (A549) and A549-ρ0. Free thiol groups in A549 cells were measured after 5 min and 8 h using Ellman's reagent. Free thiol groups in A549-ρ0 were measured after 8 h using Ellman's reagent. Data are expressed as mean values ± SD. WF-CS = exposure to 1 cigarette first passed through a receptacle of water; CS = exposure to 1 cigarette directly distributed inside the culture flasks. **P < 0.01 for CS vs. control (A549, 5 min); ##P < 0.01 for WF-CS or CS vs. control (A549, 8 h); $P < 0.05 for CS vs. control (A549-ρ0, 8 h).