Cytotoxicity of retinoic acid, menadione and aflatoxin B(1) in rat liver slices using Netwell inserts as a new culture system

Abstract

Precision-cut rat liver slices were used to develop a new dynamic incubation system in which histomorphology and measurement of the release of lactate dehydrogenase (LDH) and the conversion of MTT were applied to evaluate cytotoxicity. Liver slices, precision-cut using a Krumdieck tissue slicer, were cultured in a new system using 200-mum polyester mesh Netwell inserts in six-well cell-culture clusters on a rocker platform at 37 degrees C and 40% O(2). The major advantage of this new culture system is the easy way in which slices can be manipulated and the culture medium be sampled or changed. Rat liver slices were exposed for 4 hr to retinoic acid (RA), menadione or aflatoxin B(1) (AFB(1)). Directly after treatment and after an additional 20-hr recovery period, histomorphological observations of slices were made, and LDH release and MTT conversion were measured. Slices exposed to RA showed dose-related cytotoxicity in the MTT assay only. The cytotoxic response to AFB(1) was more pronounced in the assay of LDH release than in the MTT assay. Histomorphology, LDH release and the MTT assay revealed cytotoxic effects induced by menadione. We conclude that culturing liver slices using Netwell inserts is a good alternative to other culture systems for testing non-volatile compounds.