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Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: Activity enhancement by alamethicin, a pore-forming peptide
Citation:
Ladd, M., P. Fitzsimmons, AND J. Nichols. Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: Activity enhancement by alamethicin, a pore-forming peptide. XENOBIOTICA. Taylor & Francis, Inc., Philadelphia, PA, 46(12):1066-1075, (2016).
Impact/Purpose:
The purpose of the present study was to optimize an existing in vitro assay for hepatic UGT activity in rainbow trout. The original assay, adapted here for use with trout S9 fractions, was updated by incorporating a membrane disrupting agent (alamethicin) to reduce latency. Additional experiments were conducted to evaluate treatments (e.g., addition of Mg2+ and bovine serum albumin) that have been shown to increase UGT activity in mammalian microsomal preparations. The levels of UGT activity obtained using the “optimized” assay are substantially higher than those reported in previously published work on trout. More importantly, all reactions were conducted under physiological conditions of temperature and pH. As such, the results of this effort provide a sound basis for developing in vitro-to-in vivo extrapolation procedures for chemicals transformed by this major reaction pathway.
Description:
An existing assay for hepatic UDP-glucuronosyltransferase (UGT) activity was optimized for use with trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5’-diphosphoglucuronic acid (UDPGA; a necessary cofactor), alamethicin (a pore-forming agent added to eliminate latency), and substrate (p-nitrophenol). Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. All studies were performed under physiological conditions (pH 7.8, 11 °C) to support ongoing development of methods for extrapolating in vitro rates of biotransformation to the intact animal. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout.
