Cytotoxicity and metabolic stress induced by acetaldehyde in human intestinal LS174T goblet-like cells

Abstract

) were assessed by dichlorofluorescein, methyltetrazolium, and bioluminescence, MitoTracker green and rhod-2 doublelabeling. Membrane integrity and apoptosis were evaluated by measuring lactate dehydrogenase (LDH), caspase 3/7, and cleavage of cytokeratin 18 (CK18). Expression of mucin 2 (MUC2) was determined by cell-based ELISA. Acetaldehyde significantly increased reactive oxygen species generation and decreased mitochondrial function compared with negative controls (P Ͻ 0.05). In addition, acetaldehyde dose-dependently decreased ATP levels and induced intramitochondrial Ca 2ϩ accumulation compared with negative controls (P Ͻ 0.05). Furthermore, acetaldehyde induced LDH release and increased caspase3/7 activity and percentage of cells expressing cleaved CK18 and increased MUC2 protein expression compared with negative controls (P Ͻ 0.0001). ATP depletion and LDH release could be largely prevented by the antioxidant N-acetylcysteine, suggesting a pivotal role for oxidative stress. Our data demonstrate that acetaldehyde has distinct oxidant-dependent metabolic and cytotoxic effects on LS174T cells that can lead to induction of cellular apoptosis. These effects may contribute to acetaldehyde-induced intestinal barrier dysfunction and subsequently to liver injury.

Role of defective methylation reactions in ethanol-induced dysregulation of intestinal barrier integrity

“…It has been established that the loss of the molecular architecture of TJ which is a complex interaction of transmembrane proteins occludin, claudins with cytoplasmic adapter protein ZO-1 and a cytoskeletal network [13][14][15]52], regulates barrier function [12,[26][27][28][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. Using Caco-2 cells that exhibits this complex protein barrier, we show that ethanol exposure severely compromises the function of this barrier by affecting the localization of the major tight junction protein, occludin-1.…”

“…Despite the important role that intact intestinal barrier integrity plays in preventing organ injury, the molecular mechanism responsible for ethanol-induced gut leakiness remains an ongoing area of research [12,[26][27][28][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. It has been established that the loss of the molecular architecture of TJ which is a complex interaction of transmembrane proteins occludin, claudins with cytoplasmic adapter protein ZO-1 and a cytoskeletal network [13][14][15]52], regulates barrier function [12,[26][27][28][38][39][40][41][42][43][44][45][46][47][48][49][50][51].…”

Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation

“…Cell culture and culture medium. LS174T [CL-188 TM ; American Type Culture Collection (ATCC); Manassas, VA, USA], a human colon adenocarcinoma cell line, exhibits characteristics of mucin-secreting intestinal epithelial cells and is widely used as an intestinal goblet cell (6,19). LS174T were grown for 1 week in Eagle's minimum essential medium (EMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin.…”

“…The mucin core protein is called MUC, and more than 20 MUC types have been already identified (4). Among these MUC isoforms, mucin 2, oligomeric mucus/gel-forming (MUC2) is the main structural component of the mucus gel and represents the main secretory mucin in the small and large intestines (5,6).…”

“…Various evidences have indicated the importance of intestinal mucin (4,6,7) and its component MUC2 in maintaining intestinal homeostasis. The deficiency of mucin or MUC2 was also reported to be harmful as a result and/or a cause.…”