ABSTRACT
Title
Study of interactions between ABC-transporters and metabolism in rat intestinal "precision-cut" slices
Authors
M. Possidente
Doctorate School of Pharmacology and Toxicology, University of Siena, Italy
Doctorate School of Pharmacology and Toxicology, University of Siena, Italy
Abstract
Intestinal metabolism (phase I and/or phase II) and drug transporters (phase III) have been recognized as major physiological mechanisms that protect organisms from toxic compounds. Several phase III proteins commonly known as ATP-binding cassette (ABC) play key roles in tissue maintenance by transporting metabolic waste and toxic chemicals out of cells (Benet et al. 1999). To study xenobiotic interactions with ABC proteins like multidrug-resistance protein (MDR) and multidrug-resistance associated protein (MRP), intact cell systems are required. The aim of the present study was to set up an intestinal precision-cut slice technique to study the interaction between ABC-transporters and xenobiotic-metabolized system.
Slices were prepared as described by De Kanter et al. (2005). Slices were individually incubated in RPMI 1640 under 95% O2 5% Co2 atmosphere at 37oC in 12 well plates in presence of 0.5µM calcein AM and various concentrations of the well Known MDR or MRP inhibitors verapamil, indomethacin and glibenclamide and chlorpyrifos . The intracellular desterification product of calcein AM, calcein, was measured spectrofluorimetrically. The presence of transport inhibitors increased the intracellular concentration of calcein in a time-dependent fashion (with a maximum value at 30 or 90 min incubation time) and promoted a concentration-dependent accumulation of calcein. Furthermore verapamil, indomethacin and glibenclamide promoted a concentration-dependent accumulation of calcein (EC50 3.28 µM, 14.5 mM and 190 µM, respectively).
Furthermore the presence of 1 mM Ketoconazole, a CYP-dependent metabolism inhibitor, decreased the ABC-transporters inhibition promoted by chlorpyrifos when it was present in the medium up to 90 min incubation time. The presence of chlorpyrifos-oxon, the major cythorcome P450-depndent metabolite of chlorpyrifos, was detected in intestinal precison-cut slices by HPLC-MS analysis. These data suggested the involvement of CYP-dependent metabolism in the inhibition of ATP-transporters promoted by chlorpyrifos. Furthermore the esperiments indicated that the precision-cut intestinal slices are a reliable, simple and fast system for evaluating xenobiotic interactions with ABC transporters. Together with data already appeared in the literature (Van de Kerkhof et al. 2007), these results indicated that the model is also suitable for studying phase I and phase II drug metabolism.
References:
Benet LZ et al. (1999) Control Release 62:25-31
De Kanter et al. (2005) J Pharmacol Toxicol Methods 51:65-72
Van de Kerkhof EG et al (2007) Curr Drug Metab 8:658-75
Slices were prepared as described by De Kanter et al. (2005). Slices were individually incubated in RPMI 1640 under 95% O2 5% Co2 atmosphere at 37oC in 12 well plates in presence of 0.5µM calcein AM and various concentrations of the well Known MDR or MRP inhibitors verapamil, indomethacin and glibenclamide and chlorpyrifos . The intracellular desterification product of calcein AM, calcein, was measured spectrofluorimetrically. The presence of transport inhibitors increased the intracellular concentration of calcein in a time-dependent fashion (with a maximum value at 30 or 90 min incubation time) and promoted a concentration-dependent accumulation of calcein. Furthermore verapamil, indomethacin and glibenclamide promoted a concentration-dependent accumulation of calcein (EC50 3.28 µM, 14.5 mM and 190 µM, respectively).
Furthermore the presence of 1 mM Ketoconazole, a CYP-dependent metabolism inhibitor, decreased the ABC-transporters inhibition promoted by chlorpyrifos when it was present in the medium up to 90 min incubation time. The presence of chlorpyrifos-oxon, the major cythorcome P450-depndent metabolite of chlorpyrifos, was detected in intestinal precison-cut slices by HPLC-MS analysis. These data suggested the involvement of CYP-dependent metabolism in the inhibition of ATP-transporters promoted by chlorpyrifos. Furthermore the esperiments indicated that the precision-cut intestinal slices are a reliable, simple and fast system for evaluating xenobiotic interactions with ABC transporters. Together with data already appeared in the literature (Van de Kerkhof et al. 2007), these results indicated that the model is also suitable for studying phase I and phase II drug metabolism.
References:
Benet LZ et al. (1999) Control Release 62:25-31
De Kanter et al. (2005) J Pharmacol Toxicol Methods 51:65-72
Van de Kerkhof EG et al (2007) Curr Drug Metab 8:658-75