Cocaine Transport and Metabolism by the Colonic T84 Epithelial Cell Line 559

Abstract

To study cocaine uptake and metabolism in the colon we used polarized 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 confluency(every 7-8 days). For the experiment cells were plated on 1.0 μm collagen type I coated tissue culture inserts in 24 well plates. Cells (passages 47-55) were grown to confluency as determined by steady state trans epithelial resistance of 180 Ωcm2. 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) by GC/MS. All studies were run in duplicates. Each experiment was done in a new passage. Cocaine incubated with media alone for 30-60 min showed no metabolism. Data for cocaine and its metabolites in U and L at 60 min respectively are shown below. Results: 1) Cocaine transport across T84 cell monolayer increased linearly with increasing cocaine concentration in U. 2) Significant amounts of BE and EME were detected in U whereas only small amounts were seen in L. Conclusion: 1) Concentration dependent cocaine transport occurs across colonic monolayers. 2) Appearance of BE and EME in U in the presence, but not absence of T84 cells, suggests cellular breakdown of cocaine. 3) Significant amounts of cocaine and metabolites(37-40%) may be retained by the monolayer. Table