Abstract

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

Highlights

  • Alcohol is a major lifestyle-related risk factor for many human diseases including oral and esophageal cancers

  • Admixed with saliva and mucus produced by esophageal glands, a relatively high concentration of EtOH may stay on the esophageal mucosal surface during and after alcohol consumption; the alcohol tolerance capacity of esophageal keratinocytes remains undefined

  • Due to its physiologic location, the esophageal epithelium is vulnerable to high concentrations of EtOH exposure associated with alcohol consumption, to date there has been little evidence demonstrating the effects of alcohol on esophageal epithelial biology and homeostasis

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Summary

Introduction

Alcohol is a major lifestyle-related risk factor for many human diseases including oral and esophageal cancers. As the major site of acetaldehyde breakdown, mitochondria may produce excessive ROS when cellular acetaldehyde overwhelms the mitochondrial capacity for acetaldehyde clearance [5]; the extent to which alcoholinduced cell injury involves mitochondria in human esophageal keratinocytes remains unknown. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is the master sensor of cellular energy status. It is activated by the serine/threonine kinase LKB1 upon decreased intracellular ATP levels, which is one indicator of mitochondrial dysfunction [8]. Mitochondrial dysfunction and oxidative stress activate AMPK to promote autophagy-mediated cell survival under several established conditions including nutrient deprivation [11, 12], little is known as to how esophageal keratinocytes may survive alcohol-induced toxicity

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