Effects of ethanol and acetaldehyde on epithelial integrity in a three dimensional
(3D) epithelial cell culture model
E. Elamin1, 2, D. Jonkers 1,2 , F. Troost 1,2, K. Juuti-Uusitalo 1,3, S. van IJzendoorn 1,3,
H. Duimel 4, J. Broers 4 , F. Verheyen 4 , J. Dekker 1 & A. Masclee1,2.
1 Top
Institute Food and Nutrition (TIFN), Wageningen, The Netherlands
of Gastroenterology-Hepatology, Department of Internal Medicine, Maastricht
University Medical Center, Maastricht, The Netherlands
3 University Medical Center Groningen, Groningen, The Netherlands
4 Department of Molecular Cell Biology, Maastricht University Medical Center,
Maastricht, The Netherlands
2 Division
Introduction: A decreased intestinal barrier function and translocation of endotoxins are
involved in the pathogenesis of alcoholic liver diseases. Ethanol and its metabolite
acetaldehyde are found to disrupt tight junction structures in an in vitro two dimensional
monolayer of Caco2 cells. However, this model has limitations due to diminished
differentiation, cell signaling, and non-physiological morphology.
Aim: To investigate in a (3D) model of human colon carcinoma cells (T84), the effects of
ethanol and acetaldehyde on epithelial integrity, in concentrations of ethanol and
acetaldehyde found in the systemic and colonic circulation after ingestion of 10–30 g of
alcohol.
Methods: T84 cells were grown for 5 days to induce fully polarised spheroids. These
spheroids were then exposed to ethanol (21.7 or 43.4 mM), acetaldehyde (100 or 200
µM), culture medium (negative control) or 2 mM EGTA (positive control) for 3 hours.
Morphological differentiation was evaluated by transmission electron microscopy.
Barrier function was assessed by the flux of fluorescein isothiocyanate-conjugated
dextran 4KD (FD4) from the basal to the luminal compartment using live cell imaging.
Results: 3D cultured T84 cells form spheroids with the lumen inside, and exhibit a high
level of differentiation demonstrated by formation of microvilli and tight junctions facing
the lumen. Hence, this model is suitable to study effects of substances at the basal side.
This reflects alcohol exposure in the colon after oral intake in humans, as the transport
is characterized by a basal to luminal gradient.
After 3 hours, the mean fluorescence intensity of FD4 was measured and expressed as
the ratio of the luminal over the basal compartment, given as mean values of 7
spheroids from 4 different fields of repeated experiments. A dose-dependent increase of
the FD4 ratio was found at 21.7 and 43.4 mM ethanol versus control [0.180±0.017 and
0.436±0.008 respectively, vs 0.003±0.000, p<0.001]. Also 100 and 200 µM
acetaldehyde increased the ratio dose dependently versus control [0.203±0.038 and
0.391±0.046, respectively vs 0.003±0.000, p<0.001].
Conclusion: T84 cells cultured in 3D represent morphological features of the in vivo
intestine and are a useful model to study barrier function in vitro. Ethanol and
acetaldehyde at physiological concentrations increase intraluminal FD4 concentrations
in a dose-dependent manner, pointing to disruption of tight junctions.