|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.