Cell-Based Assays to Detect the Mechanism of Toxicity

Transcription

1 Cell-Based Assays to Detect the Mechanism of Toxicity March, 2013

2 Cell Health Assays Using Plate Readers CytoTox 96 (LDH) Apo-ONE Caspase Assay CytoTox-ONE (LDH) Caspase-Glo 3/7 CytoTox-Glo (Protease) CytoTox-Fluor (protease) CellTiter-Fluor ApoTox-Glo ApoLive-Glo CellTiter 96 (MTT) Caspase-Glo 8 & 9 MultiTox-Glo MultiTox Fluor CellTiter-Glo One Solution CellTox Green CellTiter 96 AQ ueous (MTS) CellTiter 96 AQ ueous One Soln (MTS) Colorimetric Fluorescence Bioluminescence Fluorescence & Bioluminescence CellTiter-Glo CellTiter-Blue ADME P450-Glo MAO-Glo UGT-Glo Pgp-Glo GSH-Glo GSH/GSSG-Glo Mitochondrial ToxGlo Ultra-Glo Luciferase

3 Which Assay Should I Use? First Decide What You Want to Measure Number of living cells (viability assay) Number of dead cells (cytotoxicity assay) How did the cells die Apoptosis vs. Necrosis Mitochondrial toxicity Oxidative stress Metabolic change Specific gene expression Multiplexing more than one parameter

4 Cell Health Assays Overview Viable cells detected using markers of active metabolism Cellular conversion of indicator dyes (MTT / MTS / Resazurin) Protease marker ATP content Dead cells detected using marker of membrane integrity LDH release Protease release Dye uptake / staining Apoptosis detected using caspase activities Biochemical markers of cell stress leading to cytotoxicity Mitochondrial toxicity Oxidative stress (ROS and GSH:GSSG ratio) NADH Luciferase reporters of cell stress pathways leading to cytotoxicity 5

5 Metabolic & Enzymatic Indicators of Cell Viability Reagent Tetrazolium Reagents MTT, MTS, XTT, WST Redox Indicators Resazurin Enzyme Substrates Protease Substrates Viable Cell Active Metabolism or Protease Dead Cell Loss of Function Substrate Incubation Step Product Substrate X No Rxn

6 ATP Assay for Cell Viability CellTiter-Glo Reagent Lysis Solution ATPase Inhibitors Luciferin UltraGlo Luciferase Viable Cell Dead Cell ATP Light Luciferin + Luciferase ADP X No Reaction

7 Advantages & Disadvantages of Viability Assays Assay Advantages Disadvantages MTT / MTS Resazurin Protease ATP Widely used Inexpensive Inexpensive Fluorescent readout Good sensitivity 30 min protocol Cells remain viable Better sensitivity than resazurin Good choice for multiplexing 10 min protocol Best sensitivity No fluorescence interference Lysis step stops reaction immediately (no incubation with viable cells) MTT has 2 step protocol 1-4 hour incubation Interference by reducing compounds Toxic to cells Limited sensitivity 1-4 hour incubation Interference by reducing compounds Toxic to cells Fluorescence interference Fluorescence interference Lytic protocol dictates sequence for multiplexing 8

8 Future Improvements of CellTiter-Glo Formulation CellTiter-Glo 3D Improved lytic capacity for 3D culture models CellTiter-Glo 2.0 (Abstract # TP157) Improved liquid reagent stability upon storage 10% change in performance 22⁰C 4⁰C CellTiter-Glo 12 hours 3.5 days new reagent 2 weeks 4 months* ViralTox-Glo Validated for measuring viral cytopathic effect 9

9 Balb 3T3 Cells Treated with MTT for 4 Hours Same field of cells imaged immediately after addition of MTT and after 4 hours incubation. Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences. 10

10 Balb 3T3 Cells Treated with GF-AFC for 4 Hours Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences. 13

11 Detecting Dead Cells: Two Basic Approaches The functional definition of cell viability is based on whether the outer membrane is intact. Viable Dead Dye Enzyme Marker 14

12 Cell Health Assays Overview Viable cells detected using markers of active metabolism Cellular conversion of indicator dyes (MTT / MTS / Resazurin) Protease marker ATP content Dead cells detected using marker of membrane integrity LDH release Protease release Dye uptake / staining Apoptosis detected using caspase activities Biochemical markers of cell stress leading to cytotoxicity Mitochondrial toxicity Oxidative stress (ROS and GSH:GSSG ratio) NADH Luciferase reporters of cell stress pathways leading to cytotoxicity 15

13 DNA Dye Staining to Detect Dead Cells (Overcomes some limitations of short half-life markers) Non-permeable DNA dye Staining of dead cells results in a fluorescent signal that is stable. Viable Cell X Dead Cell Dye is excluded from live cells DNA dye only stains nucleus of dead cells or debris 18

14 CellTox-Green Dye is Not Toxic to Cells ATP assay data showing viability of cells exposed to bortezomib for 72 hrs O cells exposed to DNA binding dye for 72 hr cells exposed to DNA binding dye for 15 min 19

15 HeLa Cells Treated with CellTox-Green DNA Dye for 4 Hours Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences. 20

16 Reading the Same Plate Multiple Times to Detect the Onset of Cell Death 5000 K562 cells in 96 well plate First appearance of cell death may trigger further experimentation with the same sample (e.g. How did the cells die? apoptosis?)

17 Samples with CellTox Green can be Multiplexed with Cell Viability and Apoptosis Assays 24hr

18 Triplex at each time point Endpoint multiplexes possible at first emergence of toxicity 24hr 48hr 72hr Note progression of cytotoxicity and gradual decline in caspase activity suggesting loss of enzymatic activity after release from dead cells.

19 Advantages & disadvantages of assays to detect dead cells Assay Advantages Disadvantages LDH release Widely used and accepted Absorbance or fluorescent options Limited sensitivity Limited half-life of LDH in medium Protease release Designed for multiplexing More sensitive than LDH Fluorescent reagent is simpler than formulation for LDH assay Fluorescent or luminescent options Limited half-life of protease marker Fluorescence interference (fluorescent format only) DNA Staining Non-toxic / real time assay Staining persists for 72 hours Good choice for multiplexing Fluorescence interference Less sensitive than amplified protease release assay 25

20 CellTox Green Assay: Multiplexing a Fluorescent Assay with Luminescent Assays Glo Reporter Assay Multiplexes include: Nano-Glo One-Glo Bright-Glo Steady-Glo CellTiter-Fluor Viability Assay Caspase-Glo Assays Will work Glo Reporter Will Assays Work NAD(P) / NAD(P)H- Early Access Will work CellTox Green Cytotoxicity Assay CellTiter-Glo Cell Viability Assay P450-Glo Cell-Based Assays HDAC-Glo Assay All possible with the GloMax -Multi & -Multi+ Detection Systems CytoTox-Glo Assay Will work ROS Early Access Probable BacTiter-Glo Assay GSH-Glo & GSH/GSSG-Glo Assays 26

21 Assays to Determine Cell Stress Events Leading to Toxicity

22 Determining Mechanisms Leading to Cytotoxicity Going beyond the standard assays available to detect live or dead cells. Assay chemistries and approaches to detect Apoptosis Mitochondrial toxicity Oxidative stress (ROS and GSH:GSSG ratio) Metabolic markers (NADH, NADPH) Gene expression in several stress-related pathways 28

23 Detecting Apoptosis as the Mechanism of Cell Death

24 Cell Health Assays Overview Viable cells detected using markers of active metabolism Cellular conversion of indicator dyes (MTT / MTS / Resazurin) Protease marker ATP content Dead cells detected using marker of membrane integrity LDH release Protease release Dye uptake / staining Apoptosis detected using caspase activities Biochemical markers of cell stress leading to cytotoxicity Mitochondrial toxicity Oxidative stress (ROS and GSH:GSSG ratio) NADH Luciferase reporters of cell stress pathways leading to cytotoxicity 30

25 Common Apoptosis Assays Observing morphological features TUNEL Sub G o peak of DNA using flow cytometry Annexin V binding to exposed PS (flow cytometry) Caspase-3/7 activity 31

26 AMC, R110 and aminoluciferin substrates for measuring caspase activity C H 3 C H 3 C a s p a s e 3 Z - D E V D H N O O H 2 N O O F l u o r e s c e n c e Z - D E V D H N O N H D V E D - Z H 2 N O N H 2 O C a s p a s e 3 O O O H F l u o r e s c e n c e N S C a s p a s e 3 N S Z - D E V D H N S N C O O H 2 N S N C O O L u m i n e s c e n c e L u c i f e r a s e + A T P 32

27 Luminescent Caspase Assay Caspase-Glo Reagent Lysis Solution Z-DEVD-aminoluciferin Stable Luciferase ATP Viable Cell Apoptotic Cell Dead Cell Pro-Caspase Inactive Active Caspase Inactive Caspase Reagent X No Rxn Reagent Luminescence Reagent X No Rxn

28 Lumi nescence ( Thousands) Caspase-Glo 3/7 Time Course Indicates Caspase Activity is Transient Tamoxifen Treatment of HepG2 Cells Caspase Act ivit y Cells are apoptotic at 1 hr treatment with 150µM Tamox 24 hr 6 hr 4 hr hr 1 hr hr Assay & Drug Devel Tech 2(1): 51, T a m o x i fe n (µ M ) Caspase activity decreases after 24 hours incubation

29 Luminescent Caspase-3/7 Assay Advantages: Homogeneous (add-mix-measure) X greater sensitivity than fluorescent assays No interference by fluorescent compounds Flexible incubation time to record glow signal Disadvantages: Average of entire well (not individual cells) Caspase is a transient marker Possibility of inhibition of luciferase by test compounds; but it is probably less of a problem than using a fluorescent assay

30 Detecting Mitochondrial Toxicity ATP can be used as a marker of functioning mitochondria Net ATP production from glycolysis can be blocked by using glucosefree medium* Decrease in ATP marker (without general necrosis) during 1-4hr incubation suggests mitochondrial toxicity ATP and membrane integrity assays can be multiplexed * Lisa D. Marroquin, James Hynes, James A. Dykens, Joseph D. Jamieson, and Yvonne Will. Circumventing the crabtree effect: Replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97: , X 36

31 Mitochondrial toxicity can be detected by using controlled culture conditions and short incubation Cells are exposed to treatment less than 4hr to avoid necrosis from non-mitochondrial pathways Sequential multiplex protocol is used to detect Cell death (leakage of protease marker into medium indicating loss of membrane integrity) Mitochondrial function (ATP content) Decrease in ATP without change in cell viability suggests mitochondrial toxicity 38

32 Mitochondrial ToxGlo TM Assay Multiplex membrane integrity and ATP content Change to glucose-free medium + galactose Expected Assay Profiles Treat cells 30 min - 4hr bis-aaf-r110 Substrate Incubate 30 min Record Fluorescence ATP Assay Reagent Incubate Incubate 10 min Record Luminescence No toxicity at this exposure period MitoTox Without necrosis Primary Necrosis MitoTox with necrosis

33 Oxidative Stress Assays

34 Oxidative Stress Assays Oxidative stress: an imbalance between the production of reactive oxygen species (ROS) and the cell's capacity to detoxify the ROS or to repair the oxidative damage. Markers of oxidative stress: Altered GSH:GSSG ratio (lowered GSH, increased GSSG) ROS (super oxide, hydroxyl radical, nitric oxide, hypochlorite convert to more stable H 2 O 2 ) 41

35 GSH Assay as Marker for Oxidative Stress Reduced form of glutathione (GSH) serves as an antioxidant in cells Decreased levels of GSH are associated with oxidative stress GSH and GSSG can be measured separately with a luminescent assay using Glutathione S Transferase (GST) and luciferase A fluorescent cell viability assay can be sequentially multiplexed with the luminescent GSH assay 42

36 Principal of GSH:GSSG Ratio Assay (Assays must be run in parallel in separate wells.) Total Glutathione O N S N S COOH Reduce With DTT GSSG O S O NO 2 GSH GSH CH 3 GST GS-R GSH Block with NEM GSSG Reduce GSH HO N S N S COOH Luciferase, ATP (LDR) Oxidized GSSG Light 43

37 Menadione Treatment Drops GSH:GSSG Ratio GSH:GSSG changes indicate: Oxidative Stress Compound toxicity Reactive metabolite formation

38 Reactive Oxygen Species (ROS) Assay

39 ROS-Glo H 2 O 2 Assay (coming in 2013) Direct H 2 O 2 detection without using Horseradish Peroxidase (HRP) Mitigates HRP mediated false hits Homogeneous Bioluminescent Assay Add-mix-read No fluorescence interference Cell based assay Detects H 2 O 2 content of culture wells

40 ROS-Glo Assay Chemistry Based on Pro-Luciferin Modified Pro-luciferin H 2 O 2 LDR Self-cleaving linker D-Cys cyclization Luciferase Light

41 ROS-Glo H 2 O 2 Assay Protocol Add test compound and modified pro-luciferin peroxide detector Incubate up to 2 hours ROS-Glo H 2 O 2 Assay of Hep G2 Cells Treated with Menadione Add luciferin detection reagent Incubate 15 min Record luminescence 49

42 Luminescent ROS Assay Commercial product is still in development Optional cell-based or enzymatic assay format Can sample culture medium and multiplex a cell viability assay Culture medium can affect level of ROS production Comparison of Amplex Red (requiring horseradish peroxidase) and ROS-Glo for screening LOPAC demonstrated fewer false positives with luminescent assay 51

43 Assays for NADH/NADPH, NAD +, & NADP Metabolic Indicators

44 Bioluminescent Assays for Adenine Dinucleotides Bioluminescent assays are being developed for: 1. NADH + NADPH (total of both reduced forms) 2. NAD + + NADH (total non-phosphorylated) 3. NADP + NADPH (total non-phosphorylated) Additional information on Poster # TP159 54

45 Basic Principle for the Bioluminescent Adenine Dinucleotide Detection Assays Proluciferin substrate couples NADH or NADPH to production of luciferin used to generate light Assay chemistry does not discriminate between NADH and NADPH Detects only the reduced forms NAD(P)H NA(D)P + Diaphorase Modified Luciferin Luciferin

46 Cycling Enzymes and Corresponding Substrates Provide Selectivity for Measuring Nucleotides Glucose-6-phosphate and G-6-PDH are used to measure total phosphorylated NADP + NADPH Lactate dehydrogenase and lactate are used to measure total nonphosphorylated NAD + + NADH Product G-6-P Product Lactate G-6-PDH LDH NADPH NADP + NADH NAD + Diaphorase Diaphorase Proluciferin Luciferin Proluciferin Luciferin 56

47 Cycling Enzymes and Corresponding Substrates Provide Selectivity for Measuring Nucleotides Glucose-6-phosphate and G-6-PDH are used to measure total phosphorylated NADP + NADPH Lactate dehydrogenase and lactate are used to measure total nonphosphorylated NAD + + NADH 57

48 Bioluminescent Assay Approach is More Sensitive 58

49 Summary of NAD(P) / NAD(P)H Assays Bioluminescence assay to measure NAD(P) and NAD(P)H based on the reduction of proluciferin by diaphorase One-step homogeneous 30 min cell-based assay In general, ~50X more sensitive than fluorescent assay Wide linear range; large signal window; good S/B Can measure upstream events in cancer cell metabolism that are coupled to NAD(P)/NAD(P)H production Assay NADH /NADPH NAD/NADH NADP/NADPH Application Enzyme activity assays; Direct detection of NADH & NADPH in cells Total NAD + NADH using homogenous one step assay; NAD:NADH ratio after acid/base separation Total NADP + NADPH using homogenous one step assay; NADP:NADPH ratio after acid/base separation 59

50 Cell Stress Response Pathway Reporters

51 Stress Response Pathways Leading to Cytotoxicity Stress response pathways are toxin activated signal transduction events that modulate transcription factors to trigger expression of cytoprotective genes to enable the cell to attempt to restore homeostasis.* Triggering cell response pathways occurs at lower toxin doses or exposure times than what is needed to trigger necrosis or apoptosis. If stress cannot be overcome to re-establish homeostasis, the result is induction of apoptosis and removal of the cell. *Simmons, S.O. et al., Cellular stress response pathway system as a sentinel ensemble in toxicological screening. Tox. Sci. 111(2): ,

52 Example Stress Response Pathways Oxidative Stress Response: Signaling pathway that leads to Nrf2 transcription factor binding to antioxidant response elements (ARE) that induce expression of genes to neutralize Reactive Oxygen Species (ROS) and limit oxidative damage to cellular components. (Among most commonly studies pathways) Heat Shock Response: HSF-1 activates expression of Hsp70 & Hsp27 chaperones that bind to and facilitate refolding of denatured proteins. 62

53 Skin Sensitization Model Pathway KeratinoSens Cell Line from Givaudan Modified from Fig. 2 from Natsch, A. Tox. Sci. 113(2), (2010) 63

54 Stress and Toxicity Pathway Vectors Pathway/Response Transcription Factor Name Antioxidant Nrf2 pgl4/are DNA damage p53 pgl4/p53 ER stress ATF6 pgl4/erse ER Stress ATF4 pgl4/atf4 ER stress Xbp1 pgl4/xbp1 Heavy metal stress MTF1 pgl4/mre Heat shock HSF1 pgl4/hse Hypoxia Hif1α pgl4/hre p38/jnk AP1 pgl4/ap1 Xenobiotic stress AhR pgl4/xre Inflamation NFκB Cat # E8491 Osmotic stress NFAT5 pgl4/nfat5 All constructs with pgl4.27 backbone [luc2p/minp/hygr] 64

55 Cell lines available as custom products 65

56 Questions Welcome March, 2013