Derivations of Tissue Slice Technology as Toxicological Screening Systems

Abstract

In vitro toxicology studies are hindered by the use of specific cellular systems which solely examine one cell type. Precision-cut tissue slices mimic specific organ toxicity as normal cellular heterogeneity and organ architecture are retained. Experiments were performed using tissue slices from transgenic mice with enzyme reporter proteins for rapid analysis. CYP 1A1/β-galactosidase transgenic mouse liver and kidney slices challenged with 20 μM BNF for 24 hr remain viable and display organ-specific induction of β-galactosidase (~30-fold in liver and 3-fold in kidney). AP-1/luciferase transgenic mouse tissue slices incubated with 9 μM TPA also remained viable while exhibiting a tissue- and time-dependent induction of luciferase. In kidney slices, luciferase induction was approximately 1.5-fold at 2 hr, which increased to 2.5-fold at 4 hr. Liver slices displayed a rapid increase in luciferase at 2 hr (approximately 3-fold) which was abolished at 4 hr. To quicken experimental design via decreased sample preparation, a custom transgenic mouse was created based upon a fluorescent reporter protein. Subsequent studies with slices from this strain and another fluorescent-based transgenic strain did not display reporter induction. For optimization of the smaller tissues of mice and to create an easily deployable method of rapid detection, a tissue chip based system was created for generating large numbers of samples from a single organ and coupled with fluorescent indicators to maximize detection sensitivity for specific cellular processes. Fluorescence of 5-carboxyfluorescein increased at high concentrations of iodoacetamide (IAM), a quick-acting toxicant, indicating disruption of cellular membranes. The mitochondrial probe, TMRE, exhibited an increase in fluorescence with increasing IAM concentrations. Monobromobimane, a sulfhydryl probe, displayed a decrease in fluorescent intensity at higher IAM challenge; a finding confirmed with Ellman’s reagent. A probe used for calcium measurement, FURA-2, demonstrated an increase in fluorescence with increasing IAM concentrations. Importantly, the number of samples per organ/mouse was increased at least 3-fold and a significant time reduction per analysis was realized. These results suggest that both transgenic-based tissue slice studies and studies with fluorescent probes in naïve tissue chips are two methods of higher-throughput analyses to evaluate toxicant perturbations with in vitro studies.