Characterization of Diversity in Toxicity Mechanism Using in Vitro Cytotoxicity Assays in Quantitative High Throughput Screening

Abstract

Assessing the potential health risks of environmental chemical compounds is an expensive undertaking which has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms.

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

“…A cell density of 80,000 hepatocytes/cm 2 (2K/well) was in line with HepG2 culturing conditions previously tested 19,20 and therefore was the basis for testing compound cytotoxicity in the current study for IC 50 determination as well as assessment of S/B ratio and well-to-well variation.…”

“…A 40-h exposure timepoint was extrapolated from previous publications for assessing cellular toxicity of the 12 compounds as measured by ATP content. 19,20 Functional Assessment of Cryopreserved Hepatocytes in a 1536-Well Format…”

“…The 12 compounds, a subset of the NTP 1408 library that has been used in our laboratory to validate HTS screens, have been shown to be toxic to human-and animal-derived cell lines, including liver-derived HepG2 cells. 19,20,49 To evaluate the effects of the 12 compounds, two cell densities, 2K/well and 4K/well representing 40%-50% and 80%-90% confluence, respectively, were dispensed in a 1536-well plate to assess any confluence dependent differences. The 12 compounds were tested for their cytotoxic effects on these hepatocytes after 40 h treatment and IC 50 values were calculated from the concentration-response curve for 2K/well and 4K/well conditions (Fig.…”

“…19,20 Cell lines of hepatic origin, such as HepG2 cells, have been previously adapted to HTS formats 21,22 and utilized to assess hepatotoxicity. 19,20,23 However, HepG2 cells lack the full expression of hepatocyte proteins, such as phase I and phase II metabolizing enzymes and transporters, and thus may not correlate to in vivo hepatotoxicity.…”

“…19,20 Cell lines of hepatic origin, such as HepG2 cells, have been previously adapted to HTS formats 21,22 and utilized to assess hepatotoxicity. 19,20,23 However, HepG2 cells lack the full expression of hepatocyte proteins, such as phase I and phase II metabolizing enzymes and transporters, and thus may not correlate to in vivo hepatotoxicity. [24][25][26] As an alternative to HepG2 cells, primary human hepatocytes represent the best predictive in vitro model to determine liver function for metabolism, 27,28 drug-drug interactions, 29,30 and potential hepatotoxicity of compounds.…”