Effects of cell differentiation and assay conditions on the UDP-glucuronosyltransferase activity in Caco-2 cells.

Cell differentiation increases UDP-glucuronosyltransferase (UGT) gene expression in Caco-2 cells. Glucuronidation of 13 UGT substrates, 1-naphthol, diclofenac, epitestosterone, estradiol, ethinylestradiol, indomethacin, oxazepam, R- and S-propranolol, propofol, testosterone, trifluoperazine, and zidovudine, were studied to derive a broad view on the effect of cell differentiation on the glucuronidation activities of different human UGTs. In parallel, the glucuronidation of these compounds in human liver microsomes (HLM) and human intestinal microsomes (HIM) was analyzed. Because many of the substrates are highly lipophilic, the effects of dimethyl sulfoxide (DMSO) concentrations in the reaction mixture on glucuronidation rates were tested, as well as the effect of alamethicin, a pore-forming peptide. Large differences were observed in the effects of DMSO and alamethicin between recombinant UGTs and Caco-2 cells and HLM and HIM, and, therefore, the activity assays were performed under multiple conditions. Regardless of the assay conditions, however, the results clearly indicated that although differentiation increases glucuronidation activity, the rates in Caco-2 cells are mostly very low, much lower than those in either HLM or HIM. One clear exception was observed: substrates of UGT1A6, such as 1-naphthol, were glucuronidated at very high rates in both undifferentiated and differentiated Caco-2 cells. It may thus be concluded that Caco-2 cells, even differentiated ones, do not provide a good model system to assess first-pass drug glucuronidation in the intestine.