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Alternatives to Laboratory Animals - ATLA

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Influence of Slice Thickness and Culture Conditions on the Metabolism of 7-Ethoxycoumarin in Precision-cut Rat Liver Slices


Roger J. Price, Anthony B. Renwick,1 Paula T. Barton, J. Brian Houston and Brian G. Lake

This study investigated the effects of some experimental variables on the rate of xenobiotic metabolism in precision-cut rat liver slices. Liver slices of 123 ± 8µm (mean ± SEM of six slices), 165 ± 3µm, 238 ± 6µm and 515 ± 14µm thickness were prepared from male Sprague-Dawley rats, and incubated in RPMI 1640 medium in an atmosphere of 95% O2/5% CO2 by using a dynamic organ culture system. Liver slices of all thicknesses metabolised 10µM 7-ethoxycoumarin to total (free and conjugated) 7-hydroxycoumarin in a time-dependent manner. The rate of 7-ethoxycoumarin metabolism was greatest in 165µm thick slices and slowest in 515µm thick slices, being 2.74 ± 0.19pmol/minute/mg slice protein and 0.69 ± 0.07pmol/minute/mg slice protein, respectively. No marked effects on the rate of 7-ethoxycoumarin metabolism in liver slices were observed either by changing the medium to Earle’s balanced salt solution (EBSS) or by changing the gas phase to 95% air/5% CO2. Moreover, the perfusion of rat livers with EBSS at 2–4°C, prior to preparation of tissue cores, did not enhance 7-ethoxycoumarin metabolism in rat liver slices. In this study, the optimal slice thickness was 175µm, with higher rates of 7-ethoxycoumarin metabolism being observed than with 250µm thick slices, which are often used for studies of xenobiotic metabolism. Variable results were obtained with slices of around 100–120µm thickness, which may be attributable to the ratio between intact hepatocytes and cells damaged by the slicing procedure in these very thin slices.