IN Cell User's Day 2009 講演要旨

Application of High Content Analysis For Predicting, Monitoring and Investigating Mechanisms of Toxicity

PJ O'Brien, Univ College Dublin, Ireland

The high effectiveness of HCA in testing for drug-induced, sublethal cytotoxicity is demonstrated. Cytotoxic, in vitro, biochemical and morphological effects of drugs and chemicals are concordant with potential for human toxicity across a wide range of compounds. Application of HCA in is illustrated in different cell-based models, including in vitro human HepG2 hepatoma cell lines for predictive toxicology, in vitro HuT78 human T-cell lymphoma cells, and circulating canine blood cells for clinical pathology. Such diverse applications of HCA provide some translational safety biomarkers with potential to track toxicity through drug discovery and development. HCA is also demonstrated to be useful in elucidating subcellular mechanisms of pathogenesis of drug toxicities. Critical features of cell models and effective diagnostic parameters are identified for HCA cytotoxicity testing.

High content analysis (HCA) is applied to Hep2G cells cultured in 96-well plates and loaded with fluorescent dyes for: Ca (Fluo-4 AM), mitochondrial transmembrane potential (TMRM), DNA (Hoechst 33342; to determine nuclear area and cell number) and plasma membrane permeability (TOTO-3). Cells are continuously exposed to drugs for up to 3 days at concentrations up to 100-fold the plasma concentration needed for efficacy. Compared to previous, conventional, in vitro cytotoxicity assays sensitivity is improved (~85%) while maintaining high specificity (~90%) for detection of toxic drugs.

Mechanistic information is derived. The sequence of changes in these vital parameters provides some mechanistic information on the toxicity, including mitochondrial toxicity, oxidative stress, calcium dyshomeostasis, phospholipidosis, apoptosis, and antiproliferative effects. Furthermore, the timing for the development of the toxicity provides additional information. Acutely toxic compounds are typically identified within 24 hours; but up to 3 days for chronic toxicity potential. HepG2 cells apparently are induced by the drugs they are incubated with to develop enhanced metabolic competence over the 3 days and were able to detect toxicity of all idiosyncratic hepatotoxicants studied. The phenomenon of hormesis is demonstrated, that is a biphasic response of cells to the toxicant. This occurred in ~25% compounds, and is thought to reflect an initial compensatory adaptation followed by loss of viability as this capacity is overwhelmed.

We also demonstrate that HCA can detect cytotoxicity in a human lymphoma line and in circulating blood cells from lymphomatous dogs treated with anticancer drugs. Live lymphocytes were stained as for HepG2 cells. Human T-cell lymphoma cells (HUT-78) were also treated in 96-well microtiter plates with five of 10-fold increasing concentrations of drug, starting from 0.02 uM. Concentrations producing lymphotoxicity are shown to be similar to those producing hepatotoxicity. Preliminary studies show that peripheral blood lymphocytes from lymphomatous dogs receiving chemotherapy compared to untreated dogs have abnormal viability as detected by HCA. We conclude: a) HCA can be conducted on non-adherent cells, b) dose-response relationships can be determined in vitro for lymphoma lines, and c) drug-induced cytotoxicity can detected in vivo using peripheral blood cells.