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A Real-Time, Bioluminescent Annexin V Assay for the Assessment of Apoptosis

Part # PS306

Abstract

Dose and exposure are the most important parameters used to define acute toxicological risk. However, in vitro duration of xenobiotic exposure is often relegated to convenient but arbitrary chronological endpoints. Therefore a methodological need exists to capture the kinetic detail of cytotoxic responses.A homogeneous, bioluminescent annexin V reagent was developed which can be applied to cells in plate-based formats at the time of dosing to monitor the real time progression of necrosis or apoptosis.The assay reagent utilizes two annexin fusion proteins which have been engineered to harbor separate and distinct complementing domains of a binary luciferase. These proteins respond to the kinetic emergence of phosphatidylserine (PS) exposure by proximity-based enzyme complementation. The reagent uses a time-released luciferase substrate to produce a stable bioluminescent signal proportional to PS exposure. The reagent also contains a cell impermeant, profluorescent DNA dye that measures real time loss of membrane integrity. Together, the resulting luminescent and fluorescent profiles help define cell death mechanism of action (MOA). This pilot study was conducted to examine the utility of the real time annexin reagent in a HepG2 hepatotoxicity model. Specifically, paclitaxel was employed as a control apoptosis induction agent to establish cell model response magnitudes, associated signal persistence, and to explore concordance between annexin and caspase activity biomarker measures. Compounds associated with direct or idiosyncratic hepatotoxicity (terfenadine, menadione, acetaminophen, diclofenac and aflatoxin B1) were interested to explore the kinetic relationship between potency and exposure, and MOA. Although extrapolation of in vitro toxicity data for risk assessment continues to be a challenge, we conclude that this approach enables kinetic cytotoxicity profiles that allow for rank ordering potential risk.

Printed in USA.