Early detection of toxic events induced by drug cantidats is mandatory in order to avoid late attrition in the process of R&D. Steatosis is an accumulation of triglicerids within the liver cells and may lead steatohepatiitis. Phospholipidosis is a disruption of the phospholipidases activity, either by direct inhibition or through drug-phospholipid complexes that cannot be broken by them, leading to intracellular phospholipid accumulation. Although not a toxic event by itself, phospholipidosis predict drug and metabolite accumulation that may provoke toxicity within the liver or kidney for example. Drug induced steatosis and phospholipidosis can be quantified by High Content Screening.

1. HepG2 cell model for steatosis and phospholipidosis
assessment
BURSZTYKA J.1*, ROUDAUT M.1, NDOYE A.2 & MAUBON N.1
1 HCS Pharma, 6 rue Pierre Joseph Colin, 35000 Rennes
2 Perkin Elmer , 16 av Québec, Bât Lys, 91140 Villebon sur Yvette
1*julian.burstyka@hcs-pharma.com
Abstract
Early detection of toxic events induced by drug candidates is mandatory in order to avoid late attrition in the process of R&D.
Steatosis is an accumulation of triglicerids within the liver cells and may lead steatohepatitis. Phospholipidosis is a disruption
of the phospholipidases activity, either by direct inhibition or through drug-phospholipid complexes that cannot be broken
by them, leading to intracellular phospholipid accumulation. Although not a toxic event by itself, phospholipidosis predict
drug and metabolite accumulation that may provoke toxicity within the liver or kidney for example. Drug induced steatosis
and phospholipidosis can be quantified by High Content Screening.
Results
Methods
Conclusions & Perspectives
- HepG2 cells grown in 2D are a suitable model for steatosis and phospholipidosis assay, when
incubated with well known inducers (respectively cyclosporine A and sertralin).
- This cell line is however well known for lacking phase I enzymes expression (CYP), thus raising the
question of its inability to detect active or toxic metabolites.
- Studies have found better expression or inducibility of CYP when HepG2 were grown in 3D
systems. HepG2 cells in spheroids are also able to form bile canaliculi and express MRP2
transporter.
- The next step will be to perform these assays in 3D culture.
Cell culture: HepG2 (ATCC) were grown at 37°C with 5% CO2
in MEM supplemented with 10% fœtal bovine serum,
glutmine, 1% non essential amino-acids and 1%
penicilline/streptomycine. For all assays, cells were plated at
10000 cells per well.
Cells were treated with either cyclosporin A, ketoconazole,
sertraline (0.03 to 16 µM), rifampicin, ticlopidine (0.1 to 50
µM), amiodarone (0.2 to 100 µM) or 0.5% DMSO for 48h.
Cells were stained with the HCS LipidTOXTM Phospholipidosis
and Steatosis Detection kit from ThermoFisher Scientific and
with Hoechst (DNA staining). Imaging was done on an
Operetta (Perkin Elmer) and image analysis was performed
with Columbus software (Perkin Elmer) to quantify drug
induced steatosis and phospholipidosis.
Steatosis was evaluated by detecting the spots within the
cells, and calculation of the corrected spot intensity (CSI),
that is the mean spot intensity minus its background.
Phospholipidosis was determined by calculating the mean
intensity within the cells.
Steatosis and phospholipidosis in treated cells were
compared to basal steatosis and phospholipidosis measured
in cells treated wit 0.5% DMSO alone.
Representative images of HepG2 cells
after 48h of treatment
Nuclei are stained with Hoechst (blue),
neutral lipid and phospholipidosis are
shown in green and red, respectively.
Rifampicin did not induce steatosis nor
phospholipidosis. Cyclosporin A induced
only steatosis and sertraline
phospholipidosis, whereas Amiodarone
induced both.
Cell viability, phospholipidosis and steatosis in HepG2 cells after 48h of incubation with the different compounds,
compared to control cells incubated with the vehicule alone (DMSO 0.5%, phospholipidosis and steatosis set to 1).
Dotted lines represent 3 standard deviations of the mean of the vehicle control.
Rifampicin and ticlopidine did not have any steatosis nor phospholipidosis effect.
Cyclosporin A induced only steatosis, whereas Sertraline induced only phospholipidosis.
Amiodarone and ketoconazole produced both phospholipidosis and steatosis.
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Viability
Rifampicin 25 µM Cyclosporin A 8 µM
Sertraline 2 µM Amiodarone 6.25 µM 0.5 % DMSO
Phospholipidosis Steatosis