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Abstract

Effects of Freezing on Hepatic Drug Metabolic Function Using Canine Liver Slices

John T. Gasson, BS; Maya M. Scott, BS, DVM; Dawn M. Boothe, DVM, PhD, Dip ACVIM, ACVCP

Abstract

Liver slices are an established in vitro method of examining organ toxicity and metabolism.  This technique is used with increasing frequency in university, pharmaceutical, and government laboratories.  Liver slices can be produced with reliable quality and minimal trauma, lending to their use as an alternative to in vivo toxicity and metabolism studies.  With liver slices, the normal architecture, cell heterogeneity, and cell-cell interactions of the liver are maintained.  Freshly prepared rat liver slices retain high viability for up to 48 hours of incubation and stable functions of phase I and II xenobiotic (drug metabolizing) enzyme activities.  Use of canine tissue with this methodology has been minimally studied, despite the use of dogs as models for human drug metabolism.  This study was designed to examine the impact of freezing on the viability and metabolic capacity of canine liver slices.  Successful freezing of liver slices would help maximize the use of available liver, while minimizing the number of animals needed for experimentation.  Fresh canine liver was sliced and immediate experimentation was performed with one aliquot.  The remaining aliquots were frozen at -20C in V-7, a cold preservation solution, and stored for up to 3 weeks.  Frozen slices were thawed at designated weekly intervals.  All liver slices were incubated at 37C in Waymouth's media with 10% fetal bovine serum.  Three different marker drugs-phenobarbital, lidocaine, and primidone-were added to the incubation media at varying concentrations to serve as indicators of metabolism.  Normal activity was assessed by the ability of slices to take up and metabolize drugs.  Following timed intervals of incubation of up to 24 hours, liver slices were removed and prepared for adenosine triphosphate (ATP), potassium (K+), and drug analysis.  ATP content and K+ retention were measured to assess slice viability.  Concentrations of the parent drug and its metabolites were measured in both the slice and the media to assess metabolic capacity.  Initial results suggest that freezing may have a negative impact on slice viability and drug uptake.  Compared to fresh slices, K+ retention of frozen slices was decreased.  Studies are in progress exploring the effects of freezing over time to determine maximum freezing intervals.