SOFT/TIAFT 98 - Cocaine Transport and Metabolism by the Colonic T84 Epithelial Cell Line

SOFT - TIAFT 1998

Poster Session 3

Thursday October 8, 1998

COCAINE TRANSPORT AND METABOLISM BY THE COLONIC T84 EPITHELIAL CELL LINE

Rama Bhat¹, Gopal Chari¹, Mrinalini Rao², Adam Negrusz³, Dharmapuri Vidyasagar¹

Departments of ¹Pediatrics, ²Physiology,³Pharmaceutics and Pharmacodynamics, University of Illinois at Chicago, Chicago, IL, USA

Cocaine abuse during pregnancy has a profound effect on the fetus and neonate. Cocaine permeability and metabolism in gastrointestinal tract was studied using epithelial monolayers of human colonic T84 cells. T84 monolayers were maintained in 75 cm² tissue culture flasks, in DMEM/Ham's F-12 medium containing 6 % newborn calf serum. The cells were passaged upon confluence (every 7-8 days). For the experiment, cells were plated on 1.0 mm collagen type I coated tissue culture inserts in 24 well plates. Cells (passages 47-55) were grown to confluence as determined by steady-state trans epithelial resistance of 180 Qcm².

On the day of the experiment, the culture medium from the upper (U) and lower (L) chamber was removed and replaced with serum free medium. Cocaine HCl (500-4000 ng/ml) was added to U. After incubation with cocaine, samples were obtained from both U and L at 30 and 60 min. The samples were analyzed for cocaine, benzoylecgonine (BE), norcocaine (NC) and ecgonine methyl ester (EME) using solid phase extraction and GC/MS with corresponding deuterated internal standards. All studies were run in duplicates. Each experiment was done in a new passage.

Results
Cocaine incubated with media alone for 30 and 60 min showed no metabolism. Data for cocaine and its metabolites in U and L at 60 min respectively are shown below. Results: 1) Linear increase of cocaine permeability across T84 cells was observed with increasing concentration in U. 2) Significant amounts of BE and EME were detected in U whereas only small amounts were seen in L. 3) NC was not detected.

ng added to U Cocaine (U) BE (U) EME (U) Cocaine (L) BE (L) Total conc. (U+L)

100 35.65±4.4 2.35±2.1 1.36±09 16.0±2.3 9.53 61.91±3.3

200 100.1 18.2 1.66 31.8 8.2 158.3

400 173.6±36.9 22.8±13.1 1.8 46.4±10.6 4.5±4.9 251.7±27.5

800 285.3 58.5 8.73 139.8 14.8 504.0

Conclusions

Concentration dependent cocaine transport occurs across colonic monolayers.
Appearance of BE and EME in U in the presence, but not absence of T84 cells, suggests cellular metabolism of cocaine.
Significant amounts of cocaine and metabolites (37-40%) may be retained by the monolayer.

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