A Real-Time, Bioluminescent Annexin V Assay for the Assessment of Apoptosis
Andrew Niles1, Kevin Kupcho1, John Shultz1, Wenhui Zhou2, Robin Hurst1, Jim Hartnett1, Terry Riss1, Dan Lazar1, and James Cali1
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711; 2Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401

1. Introduction

4. Kinetic Progression of Apoptosis and 2° Necrosis

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 chronologicalendpoints. 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 inplate-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 complementary subunits of a binary luciferase. These proteins respond to the kineticemergence of phosphatidylserine (PS) exposure by proximity-based enzyme complementation. Thereagent 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 thatmeasures real time loss of membrane integrity. Together, the resulting luminescent and fluorescentprofiles 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 toestablish cell model response magnitudes, associated signal persistence, and to explore concordance
between annexin and caspase activity biomarker measures. Compounds associated with direct oridiosyncratic hepatotoxicity (terfenadine, menadione, acetaminophen, diclofenac and aflatoxin B1) weretested 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, weconclude that this approach enables kinetic cytotoxicity profiles that allow for rank ordering potentialrisk.

2. Bioluminescent Annexin Assay PrincipleA.

Annexin Signal

B.

5. Concordance with an Orthogonal Apoptosis Method
Annexin Signal

Caspase Activity

B.

Exposure

Caspase EC50

Annexin EC50

15h~ 6nM~ 2nM

18h~ 6nM

~ 2nM

24h

6.5nM

2.5nM

30h

6.6nM

2.7nM

48h

4.2nM

1.9nM

Panel A. Bioluminescent annexin data were collected
in real-time using one plate with one addition. Panel B.
Caspase-3/7 activity data were collected in parallel
plates at various time points using the Caspase-Glo™
3/7 Assay in endpoint format using seven separate
plates. Panel C. Caspase activity and bioluminescentannexin EC50 values are concordant.

6. Rapid Inducers of Apoptosis
A.

Annexin Signal

Necrosis Signal

HepG2 were exposed to serial dilutions of acetaminophen or menadione for 48h as described previously.
Panel A. Acetaminophen producedPS exposure at an intermediate time period of about 8h. Potency andmagnitude of response continued to increase as a result of additional compound contact.
Panel B. Loss of membrane integrity was first evident at 12h and increased as induced cells proceeded
to the secondary necrosis phenotype.
Menadione data was similar to acetaminophen (data not shown).

8. Late Inducer of Apoptosis
A.

Annexin Signal

B.

Necrosis Signal

HepG2 were dosed with paclitaxel for 48h.

C.

3. Real-Time Workflow and Model Hepatotoxicants

Necrosis Signal

HepG2 cells were dosed with paclitaxel for 48h in the presence of the real-time Annexin V assay
reagent containing a pro-fluorescent necrosis detection probe. Luminescent and fluorescentmeasures were gathered periodically over the time course.
Panel A. Dose-dependent increases in PS exposure resulting from apoptosis emerged between
7 and 24h, with increases in the potency and magnitude of the response continuing throughout
48h. Panel B. Loss of membrane integrity was first measurable at 24h. The temporal lag
between PS exposure and loss of membrane integrity are consistent with the apoptotic phenotype.

A.

7. Intermediate Inducers of Apoptosis

Annexin Signal

B.

HepG2 were exposed to serial dilutions of aflatoxin B1 for 48h as described previously.
Panel A. Aflatoxin B1 produced PS exposure beginning at about 16h which progressed in magnitude
and potency until peaking at 28h. This late induction phenotype likely reflects the kinetic rateof biotransformation of the parent molecule by HepG2 into the more toxic and reactive epoxide form.
Panel B. Loss of membrane integrity as a result of apoptosis was first evident beginning at 24h andprogressed throughout the time course of exposure.

9. Conclusions
Necrosis Signal

The bioluminescent Annexin V assay requires one homogenous addition step
• It requires no washes or other processing steps• It can be applied to cells at the time of dosing
The assay accurately reports apoptosis in real time
• It produces substantial signal:background ratios for both PS exposure and necrosis• The real-time PS exposure data correlate with endpoint caspase activity measures

Test Agent
Paclitaxel
Acetominophen
Diclofenac
Terfenadine
Menadione
Aflaxtoxin B1

Indicated Use
Chemotherapeutic
Mild analgesic
NSAID, analgesic
Anti-histamine
Pro-vitamin1
Pro-toxin found in food stuffs

Mechanism of Toxicity
Microtuble stabilizer
Metabolite liver toxicity
Mitochondrial impairment
Arrhythmia/liver metabolism
Reactive oxygen species
Reactive epoxides

The assay can resolve the kinetics of apoptosis induction• It can define minimal and maximal exposure periods for an apoptotic response• It can characterize the relationship between potency and magnitude of response
HepG2 were dosed with serial dilutions of diclofenac or terfenadine for 48h as described previously.
Panel A. Diclofenac induced a rapid (<4h) induction of apoptosis that did not increase in potency ormagnitude as a function of additional exposure.
Panel B. Induction of apoptosis consequently produced a modest loss of membrane integrity and cell
death beginning at 16-20h.
Results with terfenadine were similar to diclofenac (data not shown).

It can be employed with various cell models exploring safety and exposure• The assay may help stratify chemical risk based on in vitro exposure• The assay may identify potency thresholds for risk assessment

1Banned in economically developed countries

March 2017www.promega.com

Corresponding author: andrew.niles@promega.com