Alcohol and its metabolites dysregulate cellular bioenergetics and induce oxidative and endoplasmic reticulum stress in primary human bronchial epithelial cells

Abstract

Background: Chronic alcohol consumption/misuse is a significant risk factor for pneumonia and lung infection leading to the development of chronic pulmonary disorders such as chronic obstructive pulmonary disease (COPD) and lung fibrosis. In this study, we sought to delineate the mechanism of alcohol-associated lung disease. We did so by measuring in vitro mitochondrial, endoplasmic reticulum (ER) oxidative stress in human bronchial epithelial cells (hBECs) treated with ethanol and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters or FAEEs) metabolites.

Methods: Primary hBECs from a normal subject were treated with relevant concentrations of ethanol and its metabolites and incubated at 37°C for 24 h. Viability and cytotoxicity were determined using cell viability and lactate dehydrogenase (LDH) assay kits, respectively. Oxidized glutathione (GSSG) and reduced glutathione (GSH) were measured by colorimetric reaction, and 4-hydroxynenonal (4HNE) by immunohistochemistry. Endoplasmic reticulum stress and dysregulated cellular bioenergetics were determined by western blot analysis. Mitochondrial stress and real-time ATP production rates were determined using a Seahorse Extracellular Flux analyzer. Amelioration of ethanol-induced oxidative/ER stress and mitochondrial energetics was determined using an AMPKα agonist.

Results: Human bronchial epithelial cells treated with ethanol, acetaldehyde, and FAEEs showed a concentration-dependent increase in the secretion of LDH, oxidative/ER stress, deactivation of AMPKα phosphorylation and mitochondrial stress (decreased spare respiratory capacity) with concomitant decreases in mitochondrial and glycolytic ATP production rates. FAEEs caused greater cytotoxicity, ER stress, and dysregulated cellular bioenergetics than those ethanol and its oxidative metabolite. AMPKα agonist-pretreated cells significantly ameliorated ethanol-induced oxidative/ER stress, deactivation of AMPKα, and dysregulated cellular bioenergetics.

Conclusions: Findings of this study suggest that ethanol and its metabolites contribute to cytotoxicity, oxidative/ER stress, and dysregulation of cellular bioenergetics in hBECs. The attenuation of ethanol-induced ER/oxidative stress and mitochondrial respiration by an AMPKα agonist may reflect a potential for it to be developed as a therapeutic agent for chronic alcohol-associated lung disease.

Keywords: AMPKα agonist; ER/oxidative/mitochondrial stress; dysregulated AMPKα signaling; ethanol; fatty acid ethyl esters (FAEE).

Fig. 1:

Cell viability (A) and Cytotoxicity (lactate dehydrogenase activity) (B) in primary human bronchial epithelial cells (hBECs) incubated with EtOH (left panel), acetaldehyde (middle panel) and FAEEs (right panel) for 24 hr. Values expressed as Mean ± SEM (n = 5 replicates). * p-value ≤ 0.05 compared to the control.

Fig. 2:

Levels of inflammatory cytokines in hBECs incubated with different concentrations of EtOH, acetaldehyde or FAEEs for 24hr. Interleukin-6 (IL-6, A), Interleukin-8 (IL-8, B), Tumor necrosis factor α (TNFα, C), Interleukin-1β (IL-1β, D), Vascular endothelial growth factor (VGEF, E), Granulocyte-colony stimulating factor (GCSF, F) and Granulocyte-macrophage colony-stimulating factor (GMCSF, G). Values expressed as Mean ± SEM (n = 5 replicates). * p-value ≤ 0.05 compared to the respective control.

Fig. 3:

Oxidized (GSSG) and reduced (GSH) glutathione ratio (marker for oxidative stress) in hBECs incubated with EtOH (left panel), acetaldehyde (middle panel) and FAEEs (right panel) for 24 hr (A). Values expressed as Mean ± SEM (n = 5 replicates). * p-value ≤ 0.05 compared to the respective control. Immunofluorescence using antibodies against 4HNE (marker for oxidative stress) (B) in hBECs incubated with EtOH (3 and 6 mg/ml), FAEEs (50 and 100 μg/ml) and acetaldehyde (2.5 and 5 μg/ml) at 37°C for 24 hours. Upper panel— DAPI; Lower panel- immunofluorescence. Relative intensity of 4HNE shown by bar diagram using Image J. Values expressed as Mean ± SEM (n = 3 replicates). * p-value ≤ 0.05 compared to the respective control.

Fig. 4:

Immunofluorescence using antibodies against GRP78 (marker for ER stress) (B) in hBECs incubated with EtOH (3 and 6 mg/ml), FAEEs (50 and 100 μg/ml) or acetaldehyde (2.5 and 5 μg/ml) at 37°C for 24 hours. Upper panel— DAPI; Lower panel- immunofluorescence. Relative intensity of GRP78 shown by bar diagram using Image J. Values expressed as Mean ± SEM (n = 3 replicates). * p-value ≤ 0.05 compared to the respective control.

Fig. 5:

Mitochondrial respiration and bioenergetics in hBECs incubated with EtOH (3 mg/ml) (A), EtOH (6 mg/ml) (B), acetaldehyde (2.5 μg/ml) (C) or FAEEs (50 μg/ml) (D) using Seahorse Extracellular Flux analyzer. Mito stress test showing mitochondrial basal oxygen consumption rate (OCR), spare respiratory capacity and mitochondrial ATP production in hBEC cells treated with EtOH, acetaldehyde and FAEEs. Data shown as the Mean ± SEM (n = 3 independent experiments). * p-value ≤ 0.05 compared to the respective control.

Fig. 5:

Mitochondrial respiration and bioenergetics in hBECs incubated with EtOH (3 mg/ml) (A), EtOH (6 mg/ml) (B), acetaldehyde (2.5 μg/ml) (C) or FAEEs (50 μg/ml) (D) using Seahorse Extracellular Flux analyzer. Mito stress test showing mitochondrial basal oxygen consumption rate (OCR), spare respiratory capacity and mitochondrial ATP production in hBEC cells treated with EtOH, acetaldehyde and FAEEs. Data shown as the Mean ± SEM (n = 3 independent experiments). * p-value ≤ 0.05 compared to the respective control.

Fig. 6:

Real-time ATP production rate in hBECs incubated with EtOH (3 mg/ml) (A), EtOH (6 mg/ml) (B), acetaldehyde (2.5 μg/ml) (C) and FAEEs (50 μg/ml) (D) using Seahorse Extracellular Flux analyzer. ATP rate assay showing total mitochondrial and glycolytic production rate for ATP production in hBECs. Data shown as the Mean ± SEM (n = 3 independent experiments). * p-value ≤ 0.05 compared to respective control.

Fig. 7.

AMPKα inactivation in hBECs incubated with different concentrations of EtOH (A), acetaldehyde (B) and FAEEs (C) for 24 hours. Representative immunoblot along with respective bar diagram showing the relative intensities of p-AMPKα/AMPKα. Intensities normalized to β-actin (loading control). Values expressed as Mean ± SEM (n = 4 replicates). * p-value ≤ 0.05 with respect to control.

Fig. 8:

Amelioration of EtOH-induced AMPKα inactivation and oxidative/ER stress in hBECs pretreated with AMPKα agonist (AICAR). Immunoblot along with respective bar diagram for p-AMPKα/AMPKα (A) in hBECs incubated with EtOH (3 mg/ml) with or without AMPKα (AICAR,1 mM) for 24 hours at 37°C. Intensities normalized to β-actin (loading control). Values expressed as Mean ± SEM (n = 4 replicates). * p -value ≤ 0.05 compared to control, and ^# p -value ≤ 0.05 with respect to EtOH. Oxidative stress as measured by GSSG: GSH ratio (B) and immunofluorescence using antibodies against 4HNE (C) in hBECs incubated with 3 mg/ml EtOH at 37°C for 24 hours in the presence of AICAR (1mM). Upper panel— DAPI; Lower panel- immunofluorescence. Relative intensities for 4HNE immunofluorescence as shown by bar diagram using Image J. Values expressed as Mean ± SEM (n = 3 replicates). * p-value ≤ 0.05 compared to control, and ^# p -value ≤ 0.05 with respect to EtOH. Immunofluorescence using antibodies against GRP78 (D), marker for ER stress, in hBECs, incubated with 3 mg/ml EtOH at 37°C for 24 hours in the presence of AICAR (1mM). Upper panel— DAPI; Lower panel- immunofluorescence. Relative intensities of immunofluorescence as shown by bar diagram using Image J. Values expressed as Mean ± SEM (n = 3 replicates). * p-value ≤ 0.05 compared to control, and ^# p -value ≤ 0.05 with respect to EtOH.

Fig 9:

Mitochondrial bioenergetics in hBECs incubated with 3 mg/ml EtOH in the presence of AICAR (1mM). Mito stress test showing baseline changes of mitochondrial respiration, spare respiratory capacity, and ATP production. Data shown as the Mean ± SEM (n = 3 independent experiments). * p-value ≤ 0.05 compared to control, and ^# p -value ≤ 0.05 with respect to EtOH.