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Mitochondrial Toxicity in Glu/Gal HepG2 Model

Mitochondrial dysfunction due to drug-induced effects, has been highlighted as a potential contributor to a number of organ toxicities including those associated with cardiac, muscular and liver systems (1).

Mitochondrial toxicity has been indicated as a contributing cause of DILI and the pre- and post-market withdrawal of several high profile pharmaceuticals as well as predominant liability in a number of pharmaceuticals with Black Box Warnings and precautionary drug labelling (2,3). Several drugs have been implicated in mitochondrial liabilities such as Amiodarone, Troglitazone, Tolcapone and Cerivastatin, which have also been indicated in other contributing DILI mechanisms (3,4).

In eukaryotic mammalian cells, mitochondria are the principal contributors for over 90 % of cellular energy in the form of ATP and are essential for both cellular metabolism and synthesis processes. Many immortalised cell models used in drug discovery in vitro studies, have metabolic adaptations for growth under non in vivo-like conditions, allowing for growth in glucose-rich media, harnessing cellular energy from glycolysis and not oxidative phosphorylation.

This process is termed the Crabtree effect (5) and has been indicated as reducing the effects of mitochondrial toxicants. By replacing glucose growth media with galactose, cellular metabolism is switched from glycolysis to oxidative phosphorylation, enhancing cellular susceptibility to mitochondrial toxicants.

Sygnature Discovery’s DMPK group assesses mitochondrial toxicity in HepG2 cell model (alternative cell lines could be available upon request).

Protocol

Compound requirements 30 mM DMSO, 100 µL
Test Article Concentrations Half log dilutions from 100 µM – 0.0003 µM, final DMSO concentration 0.3 %. (Alternative regimes available)
Incubation Time Usually test compounds are incubated for 24 hours at 37°C in a humidified CO2 tissue culture incubator followed by a cell staining procedure and absorbance measurement.
Test System  96-well microplate format – 2 x test compounds, 1 x mitochondrial toxicity control and 1 x Tox Control in duplicate, using both glucose (DMEM consisting of 25 mM Glucose) and galactose (DMEM consisting of 10 mM Galactose) media.
Analysis Method Cell viability determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) conversion to Formazan product, determined by absorbance measurement.
Controls Two mitochondrial toxicants (Rotenone and Antimycin A) and one cytotoxin (Tamoxifen) on HepG2 cell viability, grown in glucose or galactose media
Data Delivery Cell viability IC50 determined in HepG2 (Glu) and metabolism-modified HepG2 (Gal). Fold change difference measured between Glu/Gal IC50

 

Results

Figure 1. Effect of two mitochondrial toxicants (Rotenone and Antimycin A) and one cytotoxin (Tamoxifen) on HepG2 cell viability, grown in glucose or galactose media.

 

Table 1. Fold change in IC50 values when HepG2 cells are cultured in glucose and galactose, are exposed to mitochondrial toxicants.

Compound Gal EC50  (µM) Glu EC50  (µM) Glu/Gal ratio
Tamoxifen 19 ~ 26 1.4
Rotenone 0.039 8.96 230
Antimycin A 0.008 >30 >3750

 

About Us

The DMPK & Physical Sciences department at Sygnature Discovery is dedicated to understanding and optimising the absorption, distribution, metabolism and excretion of drug candidates by working in close partnership with clients and other departments within Sygnature to provide successful optimisation strategies.

We have extensive know-how and expertise to provide well validated, state-of-the-art assays and a comprehensive applied consultancy service for interpretation of the in vitro ADME and in vivo PK data.

Our corporate vision is to accelerate the discovery of new medicines, from the laboratory into development to treat patients.

Our DMPK mission is to deliver tailored DMPK expertise through innovation, quality and commitment.

References

  1. Dykens JA, Marroquin LD, Will Y. Strategies to reduce late-stage drug attrition due to mitochondrial toxicity. Expert Rev Mol Diagn. 2007 Mar 1;7(2):161–75.
  2. Labbe G, Pessayre D, Fromenty B. Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies. Fundam Clin Pharmacol. 2008 Aug;22(4):335–53.
  3. Kamalian L, Chadwick AE, Bayliss M, French NS, Monshouwer M, Snoeys J, et al. The utility of HepG2 cells to identify direct mitochondrial dysfunction in the absence of cell death. Toxicol Vitr. 2015;29(4):732–40.
  4. Dykens JA, Will Y. The significance of mitochondrial toxicity testing in drug development. Drug Discov Today. 2007;12(17-18):777–85.
  5. Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y. Circumventing the Crabtree Effect: Replacing Media Glucose with Galactose Increases Susceptibility of HepG2 Cells to Mitochondrial Toxicants. Toxicol Sci . 2007 Jun 1;97 (2 ):539–47.