検索のヘルプ

Location:ホームHot Newsトレーニング/セミナー/シンポジウム情報

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.


お問合せフォーム
※よくあるお問合せとご回答(FAQ)は「こちらで»」ご覧いただけます。
※ファイルを添付してのお問合せは、お手数ですが「Tech-JP@ge.comまでメールにて」お問合せください。
お問合せ内容[必須] お名前[必須]  

注)機種依存文字(①、② など)は使用できません。
大学/企業名[必須]
E-mail[必須]
電話[必須] - - (内線
ご記入いただく個人情報は、当社製品・サービスの提供及び販売促進、当社製品に関する情報の収集・分析及 び提供、新製品・新サービスの研究開発等並びに市場調査のために利用します。当社は個人情報を業務委託 先に預ける場合がありますが、個人情報の取扱いに関する法令、国が定める指針その他の規範に従い、委託先 に対する必要かつ適切な監督を行います。個人情報に関するお問い合わせは個人情報相談窓口(042-5855111:平日午前10時~午後5時)にて承ります。 GEヘルスケア・ジャパン株式会社 個人情報管理責任者
個人情報保護に対する基本方針