APPLICATION NOTE Profiling Drug Toxicity in Liver Cells Summary Mitochondrial toxicity is a major cause for the failure of drugs (1). Furthermore, drug-induced liver Injury (DILI) is a major health problem with a global incidence of 13.9 per 100,000, and 50% of drugs with FDA black box warnings for DILI exhibit mitochondrial toxicity. For these reasons it is important to screen drugs for mitochondrial toxicity early in the drug development process. Using the Biolog OmniLog ® System, PM-M TOX1 MicroPlates™ (Figure 1), and a new liver cell line, a simple colorimetric assay was developed to detect mitochondrial toxicity of the ox-phos uncoupler, FCCP, and the withdrawn anti-diabetic drug, troglitazone, with 8 different carbon/energy sources by measuring inhibition of tetrazolium dye reduction (2). The 8 substrates feed electrons to the mitochondrial electron transfer chain via different pathways, extending the scope and sensitivity of toxicity detection. This data plus additional data on 320 other toxic chemicals from the “ToxCast” set (3) demonstrates Biolog’s simple and highly sensitive method for detection of drug-induced mitochondrial toxicity. Results LW5-3 liver cells, a HepG2 derivative, in RPMI 1640 medium (without glucose) were dispensed into wells of the Biolog PM-M TOX1 MicroPlates whose rows contain different carbon/energy sources: glucose, inosine, galactose, glucose- 1-phosphate, xylitol, α-ketoglutarate, β-hydroxybutyrate, and pyruvate. After a one day pre-incubation, 2-fold serial dilutions of FCCP or Troglitazone were titrated across the rows of the TOX1 MicroPlates. After two more days of incubation with the drugs, Biolog Redox Dye Mix MB containing glucose was added and the rate of the tetrazolium reduction was measured with a Biolog OmniLog ® automated incubator-reader over an 18 hr period (Figures 2, 3, 4a & 4b). Conclusions This study with Biolog’s PM-M TOX1 MicroPlates demonstrates a simple and highly sensitive assay technique to detect and categorize chemicals that are cytotoxic, or more specifically, those that exhibit mitochondrial toxicity. Using a proprietary redox dye chemistry that gives a simple colorimetric readout and a novel liver cell line with greater metabolic versatility than HepG2, one can profile the response of mitochondrial energy generation with 8 diverse carbon energy/substrates. Use of the OmniLog® Phenotype MicroArray platform and kinetic analysis software to incubate read and record 50 MicroPlates at a time permits accurate quantitation of metabolic rates for detecting drug-induced mitochondrial toxicity on 8 energy pathways simultaneously. Figure 1: PM-M TOX1 MicroPlate. The MicroPlate has 96 wells. Each of the 8 rows is coated with a different oxidizable carbon source that can be metabolized by most mammalian cells to produce energy. The 12 columns of the MicroPlate can be used for testing a chemical over a 1000-fold concentration range. Figure 2: The OmniLog PM-M System. The system offers automated incubation, data collection and data processing for 50 plates, with true “random access” convenience for multiple users. ¹ Will, Y., Dykens, J. Mitochondrial toxicity assessment in industry – a decade of technology development and insight. Expert Opin Drug Metab Toxicol. 2014 Aug:10(8):1061-7. doi: 10.1517/17425255.2014.939628. ² Wiater, L., Noble, S., Bochner, B., Profiling Toxic Chemicals with a New Liver Cell- based Assay, Poster presented at ASSLD, 2011. 3 ToxCast: http://epa.gov/ncct/toxcast/ 00A 054 Rev A 1/2015
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A P P L I C A T I O N N O T E
Profiling Drug Toxicity in Liver CellsSummary
Mitochondrial toxicity is a major cause for the failure of drugs (1).Furthermore, drug-induced liver Injury (DILI) is a major healthproblem with a global incidence of 13.9 per 100,000, and 50% ofdrugs with FDA black box warnings for DILI exhibit mitochondrialtoxicity. For these reasons it is important to screen drugs formitochondrial toxicity early in the drug development process.
Using the Biolog OmniLog® System, PM-M TOX1 MicroPlates™(Figure 1), and a new liver cell line, a simple colorimetric assaywas developed to detect mitochondrial toxicity of the ox-phosuncoupler, FCCP, and the withdrawn anti-diabetic drug,troglitazone, with 8 different carbon/energy sources by measuringinhibition of tetrazolium dye reduction (2). The 8 substrates feedelectrons to the mitochondrial electron transfer chain via differentpathways, extending the scope and sensitivity of toxicity detection.
This data plus additional data on 320 other toxic chemicalsfrom the “ToxCast” set (3) demonstrates Biolog’s simple andhighly sensitive method for detection of drug-inducedmitochondrial toxicity.
Results
LW5-3 liver cells, a HepG2 derivative, in RPMI 1640 medium(without glucose) were dispensed into wells of the Biolog PM-MTOX1 MicroPlates whose rows contain differentcarbon/energy sources: glucose, inosine, galactose, glucose-1-phosphate, xylitol, α-ketoglutarate, β-hydroxybutyrate,and pyruvate. After a one day pre-incubation, 2-fold serialdilutions of FCCP or Troglitazone were titrated across therows of the TOX1 MicroPlates. After two more days ofincubation with the drugs, Biolog Redox Dye Mix MBcontaining glucose was added and the rate of the tetrazoliumreduction was measured with a Biolog OmniLog® automatedincubator-reader over an 18 hr period (Figures 2, 3, 4a & 4b).
Conclusions
This study with Biolog’s PM-M TOX1 MicroPlates demonstratesa simple and highly sensitive assay technique to detect andcategorize chemicals that are cytotoxic, or more specifically,those that exhibit mitochondrial toxicity. Using a proprietaryredox dye chemistry that gives a simple colorimetric readoutand a novel liver cell line with greater metabolic versatility thanHepG2, one can profile the response of mitochondrial energygeneration with 8 diverse carbon energy/substrates.
Use of the OmniLog® Phenotype MicroArray platform andkinetic analysis software to incubate read and record 50MicroPlates at a time permits accurate quantitation of metabolicrates for detecting drug-induced mitochondrial toxicity on 8energy pathways simultaneously.
Figure 1: PM-M TOX1 MicroPlate. The MicroPlate has 96 wells.Each of the 8 rows is coated with a different oxidizable carbon sourcethat can be metabolized by most mammalian cells to produce energy.The 12 columns of the MicroPlate can be used for testing a chemicalover a 1000-fold concentration range.
Figure 2: The OmniLog PM-M System. The system offers automatedincubation, data collection and data processing for 50 plates, with true“random access” convenience for multiple users.
¹ Will, Y., Dykens, J. Mitochondrial toxicity assessment in industry – a decade oftechnology development and insight. Expert Opin Drug Metab Toxicol. 2014Aug:10(8):1061-7. doi: 10.1517/17425255.2014.939628.
² Wiater, L., Noble, S., Bochner, B., Profiling Toxic Chemicals with a New Liver Cell-based Assay, Poster presented at ASSLD, 2011.
3 ToxCast: http://epa.gov/ncct/toxcast/
00A 054 Rev A 1/2015
Figure 3: Troglitazone dose inhibition of Biolog Redox Dye MB reduction by LW5-3 cells after 2 day exposure to drug, in Biolog PM-M TOX1 MicroPlates. Assayed in triplicate.
Figure 4a: FCCP dose inhibition of Biolog Redox Dye MB reduction by LW5-3 cells after 2 dayexposure to drug, in Biolog PM-M TOX1 MicroPlates. Assayed in triplicate.
Figure 4b: Biolog Redox Dye Mix MB reduction by LW5-3 liver cells incubated in IF-M1 + 2mMGln + PS + indicated carbon source after exposure to FCCP over the concentration range shown.Pyruvate provides for more sensitive detection of FCCP toxicity than glucose or other substrates.
Carbon/Energy Source
Glucose
Inosine
Galactose
Glucose-1-Phosphate
Xylitol
α-ketoglutarateβ-hydroxybutyrate
Pyruvate
[FCCP] /uM
0 0.1 0.2 0.4 0.8 1.6 3.1 6.25 12.5 25 50 100
Carbon/Energy Source
Glucose
Inosine
Galactose
Glucose-1-Phosphate
Xylitol
α-ketoglutarateβ-hydroxybutyrate
Pyruvate
[Troglitazone] /uM
0 0.1 0.2 0.4 0.8 1.6 3.1 6.25 12.5 25 50 100
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