Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies. I. Glucose metabolism and enzyme release as cytotoxic markers

Abstract

Cytotoxicity endpoints, spontaneous glucose secretion/consumption and LDH and γ-GT release, were characterised in rat liver and HepG2 spheroids as in vitro models for toxicology studies. Preprepared rat liver spheroids and HepG2 spheroids cultured in a six-well plate format were exposed to varying concentrations of galactosamine, propranolol, diclofenac, and paracetamol. All four model toxins significantly affected glucose secretion, which agreed well with LDH and/or γ-GT release in rat liver spheroids. These toxins also significantly increased LDH and/or γ-GT release in HepG2 spheroids. Whereas glucose consumption in HepG2 spheroids did not show conclusive results, LDH activities in both types of spheroids were similar and their levels were relatively high. Accordingly, the level of LDH leakage in both types of spheroids was much higher than γ-GT after exposure to the toxins. In contrast, γ-GT activity in HepG2 spheroids was sixfold higher than that in rat liver spheroids. This study revealed that galactosamine interfered with the γ-GT assay and paracetamol interfered with the LDH assay. It demonstrated, for the first time, that glucose secretion by liver spheroids can be used as a functional indicator of cytotoxicity. Test compounds may interfere with enzymatic assays as indicated by LDH and γ-GT release in this study. Combining functional parameters together with two or more indicators of enzyme releases can provide a reliable cytotoxicity evaluation. Liver and HepG2 spheroids as in vitro models showed good predictions in chemical-induced hepatic cytotoxicity.