Editor's Highlight: Mechanistic Toxicity Tests Based on an Adverse Outcome Pathway Network for Hepatic Steatosis

Abstract

Risk assessors use liver endpoints in rodent toxicology studies to assess the safety of chemical exposures. Yet, rodent endpoints may not accurately reflect human responses. For this reason and others, human-based invitro models are being developed and anchored to adverse outcome pathways to better predict adverse human health outcomes. Here, a networked adverse outcome pathway-guided selection of biology-based assays for lipid uptake, lipid efflux, fatty acid oxidation, and lipid accumulation were developed. These assays were evaluated in a metabolically competent human hepatocyte cell model (HepaRG) exposed to compounds known to cause steatosis (amiodarone, cyclosporine A, and T0901317) or activate lipid metabolism pathways (troglitazone, Wyeth-14,643, and 22(R)-hydroxycholesterol). All of the chemicals activated at least one assay, however, only T0901317 and cyclosporin A dose-dependently increased lipid accumulation. T0901317 and cyclosporin A increased fatty acid uptake, decreased lipid efflux (inferred from apolipoprotein B100 levels), and increased fatty acid synthase protein levels. Using this biologically-based evaluation of key events regulating hepatic lipid levels, we demonstrated dysregulation of compensatory pathways that normally balance hepatic lipid levels. This approach may provide biological plausibility and data needed to increase confidence in linking invitro-based measurements to chemical effects on adverse human health outcomes.

Keywords: adverse outcome pathway; chemical risk assessment; hepatic steatosis; high-throughput toxicity testing; mechanistic toxicology; nonalcoholic fatty liver disease.

Figure 1.

Representative images of HepaRG cells treated with Wyeth-14,643 (50, 15.8, 5.0 μM), T0901317 (15.8, 5.0, 1.58 μM), troglitazone (10.0, 3.16, 1.0 μM), cyclosporin A (50, 15.8, 5.0 μM), 22(R)hydroxycholesterol (50, 15.8, 5.0 μM), amiodarone (50, 15.8, 5.0 μM), DMSO, and untreated. Fluorescence of Nile Red (to selectively stain for phospholipid rich environments (red) or lipid droplets (yellow); and Hoechst 33342 to stain for nuclei (blue) are included, 20× magnification.

Figure 2.

Key biological event assessment in HepaRG cells utilizing assays for lipid droplet accumulation (A), phospholipid accumulation (B), fatty acid uptake (C), and fatty acid efflux (D). HepaRG cells were treated for 48hours with 1/2 log dilutions of 22(R)hydroxycholesterol, amiodarone, cyclosporine A, T0901317, troglitazone, Wyeth-14,643, and DMSO control. Lipid accumulation (A,B), fatty acid uptake (C), and APOB100 levels (fatty acid efflux, D) were assessed. Data are presented as the log₂ FC of test samples and were considered significant for log₂ FC ≥ 3 times the BMAD of DMSO controls (threshold). Bars are standard error, data points are median value, and data in the shaded region are within the threshold, reported from n=3–4 technical replicates.

Figure 3.

FAO was assessed in HepaRG cells treated with test compound (15.8 μM Wyeth-14,643, T0901317, cyclosporin A, 22(R)hydroxycholesterol, amiodarone, 10 μM troglitazone, 40 μM etomixir [negative control], and 0.01% DMSO) using the Seahorse Flux Analyzer XFe96 FAO Assay. The results are shown for chemical effects on OCR due to the oxidation of the exogenous fatty acid palmitate. Test compound, oligomycin, FCCP, and a mix of rotenone and AA were serially injected to measure chemical effects on adenosine triphosphate production, maximal respiration, and nonmitochondrial respiration. The data were baselined to the median rotenone/AA measurement at time point 15 of DMSO controls and scaled from 0 to 100. The basal threshold at T6 (after chemical injection) and maximal response at T12 (after FCCP injection) were analyzed to identify chemical effects on mitochondrial respiration outside of the response threshold (shaded region). Data reported from n=4 technical replicates.

Figure 4.

FASN protein levels were assessed from cyclosporin A and T0901317 treated HepaRG cell homogenate by an automated capillary immunoassay system. Bars represent FC relative to DMSO controls (dashed line). * for p < .05 by Mann Whitney test, n=3.

Figure 5.

Summary of an AOP-based toxicity test for steatosis. An AOP network for hepatic steatosis was used to develop mechanistic toxicity tests for KEs. Leveraging scientific knowledge, mechanistic AOP-based toxicity tests, and dosimetry, the community may be able to rapidly and formally describe (with empirical data) causal relationships between exposure and health effects.