Low concentrations of ethanol induce apoptosis in human intestinal cells.
BACKGROUND
Increased systemic levels of endotoxin have been detected in human alcoholics and are thought to be derived from the gut. Although a 'leaky gut' is considered to be a necessary factor for alcohol-induced endotoxemia followed by chronic liver injury, the effects of low concentrations of ethanol on intestinal epithelial cells have not been fully understood. The aim of this study was to evaluate intestinal epithelial cell death induced by acute, low concentrations of ethanol in an in vitro system.


METHODS
The human intestinal Caco-2 cell line was incubated with 0%, 5%, 10% ethanol for up to 3 h. Phosphatidylserine (PS) externalization, caspase-mediated cytokeratin 18 (CK18) cleavage, and DNA fragmentation were evaluated using flow cytometry. The caspase inhibitor zVAD-fmk was used to test the role of caspases in ethanol-induced cell death.


RESULTS
Treatment with 5% and 10% ethanol for 3 h led to a gradual increase in PS externalization. Caspase-mediated CK18 was significantly enhanced as early as 1 h after 10% ethanol incubation, while DNA fragmentation was detected from 2 h onwards. Not only caspase activation but also both PS externalization and DNA fragmentation were completely prevented by pretreatment with the caspase inhibitor.


CONCLUSIONS
Apoptotic cell death in confluent Caco-2 cells was induced by acute and low concentrations of ethanol. These results suggest that clinically achievable doses of ethanol impair intestinal barrier function by induction of apoptosis in intestinal epithelial cells. This impairment of the barrier function would allow endotoxin to enter the circulation and evoke hepatic inflammation.
Asai
K
K
Dept. of Surgery, University of Maastricht, The Netherlands.
Buurman
W A
WA
Reutelingsperger
C P M
CP
Schutte
B
B
Kaminishi
M
M
D000230:Adenocarcinoma / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D018938:Caco-2 Cells / Q000187:drug effects / Q000378:metabolism; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology; D000431:Ethanol / Q000008:administration & dosage*; D006801:Humans; D007422:Intestines / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D010718:Phosphatidylserines / Q000378:metabolism; D012997:Solvents / Q000008:administration & dosage*; D014407:Tumor Cells, Cultured
D010718:Phosphatidylserines; D012997:Solvents; D000431:Ethanol; D020169:Caspases