Effects of Cell Differentiation and Assay Conditions on the UDP-Glucuronosyltransferase Activity in Caco-2 Cells

Abstract

ABSTRACT: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.

UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of Bovine Serum Albumin

“…The solid residues were dissolved in the reaction mixture that was composed of phosphate buffer (50 mM, pH 7.4), MgCl 2 (10 mM), BSA (0 or 0.1%), and the enzyme source (0.02-0.2 mg/ml total membrane protein, either UGT1A9-enriched or Bac control membranes), to a final volume of 100 l. The concentration of MgCl 2 required for optimal UGT1A9 activity was selected based on literature data (Walsky et al, 2012). Alamethicin was not added because it does not significantly stimulate the glucuronidation activity of recombinant UGT enzymes expressed in Sf9 insect cells (Kaivosaari et al, 2008;Zhang et al, 2011;Walsky et al, 2012).…”

Bovine Serum Albumin Decreases Km Values of Human UDP-Glucuronosyltransferases 1A9 and 2B7 and Increases Vmax Values of UGT1A9

“…Alamethicin was not added to the incubations with recombinant UGTs because it has no significant effect on the glucuronidation activity of such samples (Zhang et al, 2011).…”

Characterization of Hepatic and Intestinal Glucuronidation of Magnolol: Application of the Relative Activity Factor Approach to Decipher the Contributions of Multiple UDP-Glucuronosyltransferase Isoforms

“…The system consisted of a Waters Acquity ultraperformance liquid chromatograph equipped with an Acquity BEH C18 column (1.7 m, 2.1 ϫ 100 mm) and an Acquity PDA detector and a Waters Xevo Q-Tof mass spectrometer. Mass spectrometry was performed in negative ion mode from m/z 100 to 1000, as described previously (Zhang et al, 2011). Dopamine glucuronidation was analyzed using a Waters Acquity ultraperformance liquid chromatograph and an Agilent 6410 triple-quadrupole mass spectrometer, as described previously .…”