In vitro models of hepatotoxicity

1. In vitro models of hepatotoxicity
Authors: Tea Omanović, Vjera Ninčević and Martina Smolić
Affiliation: Department of Pharmacology, Faculty of Medicine Osijek, University of Osijek,
J. Huttlera 4, 31000 Osijek, Croatia
1. Materials and methods used to study hepatotoxicity in vitro
1.1. Hepatocyte cell cultures
Two cell lines with different characteristics will be used for in vitro models of hepatotoxicity:
1. HepG2 cells are derived from a liver hepatocellular carcinoma of a 15 year old Caucasian
male and retain many characteristics of normal differentiated quiescent hepatocytes (1).
2. HuH7 cells are well differentiated hepatocytes derived from cellular carcinoma cell line
that was originally taken from a liver tumor in a 57-year-old Japanese male in 1982.
Above mentioned cell cultures are incubated with DMEM (Dulbecco's Modified Eagle Medium)
+ 10% FBS (fetal bovine serum) + 1% pen-strep (penicillin-streptomycin), in a humidified
incubator supplied with 5% CO2 at37 °C. Medium should be changed twice a week.
1.2. The Trypan Blue dye exclusion test
Cells viability in a suspension can be determined by the Trypan Blue dye exclusion test. Live cells
possess intact cell membranes that exclude certain dyes, among all trypan blue, whereas dead cells
do not (2). A cell suspension (if cells were cultured on plates, they should be detached prior to this
test) is simply mixed with the dye (eg. 10µl of cell suspension and 10µl of dye). Cells are counted
using hemocytometer (device that consists of a thick glass microscope slide with a rectangular
indentation that creates a chamber). Prior to using the hemocytometer, first make sure that the
special coverslip is properly positioned on the surface of the counting chamber. Apply cell
suspension to the edge of the coverslip to be sucked into the void by capillary action which
completely fills the chamber with the suspension. Afterwards, visually examine to determine if
cells take up or exclude dye. Clear cytoplasm will be present in viable cells whereas dead cells
will have a blue cytoplasm. Hemocytometer should be placed on the stage of a light microscope
and focus should be onto the cells. There are different methods of counting the cells, one of them
is to count just the viable cells with clear cytoplasm and then use this equation:

2. 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
𝑚𝑙
= 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×
𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠
× 10 000
𝑐𝑒𝑙𝑙𝑠
𝑚𝑙
1.3. Oil red O staining
Oil red O is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and
lipids in different tissue samples. It has the appearance of a red powder with maximum absorption
at 518 (359) nm. 4% paraformaldehyde (cooled at 4°C) is used for cells fixation. After fixing the
cells for 30 - 45 min at 4 °C, remove fixative and rinse twice with cooled phosphate buffered saline
(PBS). Remove PBS and allow to air dry. Meantime, mix Oil red O stock (0.5% Oil Red O stock
consists of 0,5g Oil Red O diluted in 100ml of 99% isopropanol) at 6:4 ratio with distilled H2O
(dH2O) and let stand for 10 min. The working solution is stable for no longer than 2 hours, so
make up only what will be used in that time. Use a 0.2 micron syringe filter to add Oil red O to
cells. If the Oil Red O is not filtered properly you will have a lot of background staining. Slowly
rotate the dish to spread Oil Red O evenly over the cells. Leave the Oil red O for 10 min then
remove and rinse well with dH2O until the water runs clear. Add hematoxylin counterstain into
the well so that the cells are completely covered and let stand for 1 minute. Then remove and rinse
well with dH2O until the water runs clear. Leave the final dH2O rinse on the cells for microscopy
and analysis. Plates should be viewed on a phase contrast microscope where lipids will appear red
and the nuclei will appear blue.
1.4. MTT
The MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay is a colorimetric
assay for evaluating cell metabolic activity and proliferation. Conversely, when metabolic events
lead to apoptosis or necrosis, the reduction in cell viability can be determined by MTT. The yellow
tetrazolium MTT is reduced by metabolically active cells, in part by the action of dehydrogenase
enzymes, to generate reducing equivalents such as NADH and NADPH (3). The resulting
intracellular purple formazan can be solubilized and the absorbance of this colored solution can be
quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a
spectrophotometer (3). MTT is suitable for investigation of drug hepatotoxicity, considering that
drug hepatotoxicity investigation often requires testing several different concentrations and drug
exposure times using cells in culture (4). Accordingly, it is attractive to use a viability test that
allows the analysis of many samples with little handling time, such as MTT (4). MTT assays should

3. be done in the dark since the MTT reagent is sensitive to light. First step is to plate cells, usually
in 96-well plate (e.g. 2x103 cells per well for a week long experiment) and incubate for 6-24 hours.
Next day treat cells with agents for appropriate time. Afterwards, remove medium and add fresh
medium with 0.5 mg/ml MTT. Incubate for 4-8 hours (depending of cell line, cell density etc.; e.g.
6 h for Huh7, Huh7.5 and 3T3 cells), until purple precipitate is clearly visible Remove MTT media.
Add equal volume of 0.04 M HCl in isopropanol or 10% SDS solution to solubilize cells. (3).
Read absorbance at 595 nm on Microplate reader. Absorbance values that are lower comparing to
the control cells signify a reduction in the rate of cell proliferation, as opposed to higher absorbance
rates which indicate an increase in cell proliferation (3).
1.5. RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR), represents variant of PCR preceded
with conversion of sample RNA into cDNA. This technique is frequently used in molecular
biology to detect gene expression through creation of cDNA transcripts from RNA using enzyme
reverse transcriptase. (5). Afterwards, amplification of the newly synthesized cDNA is done by
traditional PCR. First step of this method represents reverse transcription. Template RNA (1-10
µg), primers (1µl random oligos) and DEPC-H2O (DNase-RNase- free water) up to 14µl should
be mixed together in PCR tube, incubated at 70°C for 10 minutes, and cooled on ice. Afterwards,
add 7.8-8µl of reaction mix (2µl 10xPCR buffer, 1µl 50mM MgCl2, 1µl 10mM dNTPs, 2µl 0.1M
DTT (dithiothreitol), 1µl DEPC-H2O and 0.5-1µl reverse transcriptase) to each sample and
incubate 50 minutes at 42°C. Stop reaction by heating to 70°C for 15 minutes and cool on ice.
Subsequently, add 1µl RNase inhibitor (10µg) to each sample and incubate 20 minutes at 37°C.
At this moment, cDNA samples can be used for standard PCR (second step), or they can be stored
at 20°C for longer period of time. Second step begins by adding primers (10µl each) to 0.5 PCR
(0.5ml) tune. Create master mix of reagents, which consists of (for 100µl reaction): 10xPCR buffer
(10µl), 10mM dNTPs (2µl), 50mM MgCl2 (3µl) and Taq polymerase (0.5µl). Add 15.5µl of master
mix to each sample. Subsequently, add 1-10mg dilution of RT-PCR cDNA and DEPC-H2O up to
65µl. Overlay each sample with 3 drops of mineral oil. Finally, run samples (should be 100µl total)
in PCR cycle.
1.6. Western blotting
Western blotting is a widely used technique which enables detection of specific proteins in a
complex mixture of proteins extracted from cells (6). This technique can be divided into three
major steps: separation of proteins by size (gel electrophoresis), electrotransfer to a solid support
and finally blocking and antibody incubation (6). First step includes standard sample and gel

4. preparation preceding SDS-PAGE electrophoresis. Second step represents electrotransfer, which
makes proteins accessible to antibody detection by moving them from within the gel onto a
membrane made of nitrocellulose or polyvinylidene difluoride (PVDF) membrane (6).
Electroblotting uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose
membrane (proteins are negatively charged, therefore be sure to place membrane between the gel
and anode). Filter sheets and membrane should be cut to fit the measurement of the gel. Wet the
membrane in MetOH and the sponge and filter paper in transfer buffer. Afterwards create a transfer
sandwich which includes gel, membrane, a fiber pad (sponge) at each end, and filter papers to
protect the gel and blotting membrane. Move the sandwich to transfer apparatus and add transfer
buffer to the apparatus. Then place the electrodes and transfer for 45-90 minutes (depending on
the thickness of the gel). Last step begins by blocking the membrane (prevents non-specific
background binding of the antibodies) with 5% skim milk in TBST (Tris-Buffered Salin Tween-
20) for 1 hour. Afterwards, add primary antibody in 5% BSA and incubate (the incubation time
can vary between a few hours and overnight, depending on the binding affinity of the antibody)
on shaker. Wash the membrane 3 times with TBST for 5 minutes and then incubate with the
recommended dilution of conjugated secondary antibody in blocking buffer at room temperature
for 1 -2 hours. Agitation of the antibody is recommended to enable adequate homogenous covering
of the membrane and prevent uneven binding. Repeat the washing procedure. The secondary
antibody is usually labeled to a reporter enzyme such as horseradish peroxidase (HRP). The
membrane is therefore detected by the signal that labeled antibody (HRP plus enzyme substrate)
produces corresponding to the target protein position. Finally, by developing a film in a dark room,
signal is captured.
2. Fatty acids- in vitro models of NAFLD
Fatty acids (FFA), more precisely, palmitic (PA) and oleic acid (OA) represent determinants of
the pathophysiology of NAFLD (1). Palmitic and oleic acid are the most widely distributed
saturated and monounsaturated fatty acids in nature. Various studies have demonstrated that PA
and OA mixtures-induced steatosis is associated with lipotoxicity, liver injury, apoptosis and
steatosis in hepatocyte cell cultures (7). The amount of fat accumulation due to fatty acids activity
is comparable to that observed in livers of patients with NAFLD (8). Therefore, it is important to
master the knowledge and skills needed to develop in vitro models of NAFLD using free fatty
acids for a better understanding of disease itself.

5. 2.1. Fatty acids
Long-chain FAs, palmitic (16:0) and oleic (18:1) are provided as sodium salts. PA and OA are
dissolved in MetOH 99% (stock solution 100 mM). Stock solutions are kept at -20°C before the
experiments.
2.2. Protocol of the study—induction and evaluation of steatosis
HepG2 and HuH-7 cell cultures are incubated with above mentioned medium supplemented with
FA at the following final concentrations: a) PA: 0.33 mM and 0.66 mM; b) OA: 0.66 mM and 1.32
mM (1). Incubate control cell cultures with plain medium and with medium added with the vehicle
in which fatty acids were dissolved (in this case MetOH 99%). Figure 2.
Figure 2. 6-well plate with cell culture tested with FFAs as mentioned in text
1- Plain medium
2- Medium plus MetOH 99%
3- Medium plus 0.33mM PA solution
4- Medium plus 0.66mM PA solution
5- Medium plus 0.66mM OA solution
6- Medium plus 1.32mM OA solution
Various studies showed that optimal incubation period for getting the expected outcomes is 24h
hours (when incubating for 12h or less the triglyceride accumulation is low, following undetectable
variations in gene expression, in contrast, longer incubation period doesn’t provide a significant
advantage in terms of intracellular triglyceride accumulation, but significantly decreases cell
viability at higher FA concentrations) (1). Next step includes measurement of the extent of
steatosis, apoptosis and gene expression. Steatosis is measured by previously explained method
Oil-Red O staining (1.3), where lipid droplets should be colored in red, whereas nuclei in blue.
Level of apoptosis is determined by MTT (1.4). PA generally has higher ability to induce apoptosis
in cell cultures, compared to OA. Various studies use rabbit Phospho-Akt Antibody and Anti-β-
Actin antibody as primary antibodies for Western blot analyses. Finally, RT-PCR is used for
1 2 3
4 5 6

6. demonstrating gene expression. Expression of three genes has been found constantly changed after
treating cells with FAs: PPARα, PPARγ and SREBP-1 (1).
3. Acetaminophen induced hepatotoxicity
Acetaminophen (APAP) represents the most often used analgesic and antipyretic drug in general.
Nevertheless, hepatotoxicity caused by APAP overdose is the most common cause of acute liver
failure worldwide (9). To investigate all the molecular mechanisms involved in APAP
hepatotoxicity, various methods have to be used. Different studies offer in vitro approach to this
subject as one of the most effective ones. Primarily, incubate hepatic cell cultures in medium
containing APAP (0.5, 10, or 20 mM dissolved in 100% of dimethyl sulfoxide) for 2, 6, and 24 h
(10). To investigate the viability of hepatocytes through the mitochondrial function, the MTT assay
is used as previously described (9). Pparα, Faah, Nape-pld Mcp1, Tnfα, and Il6 genes represent
targets for measurement of gene expression by RT-PCR (10). Antibodies recommended for use
for protein expression by western blotting are PPARα, NAPE-PLD and FAAH. APAP which was
acutely and repeatedly administered to induce liver injury, modifies the expressions of the NAE-
based anti-inflammatory system composed by the PPARα, the NAEs synthesis enzyme NAPE-
PLD and the NAEs degradation enzyme FAAH. Significant decrease in expression of above
mentioned proteins was demonstrated 6h after treatment with APAP (10) . Interestingly, this effect
on proteins expression was completely reversed after 24 h from acute APAP administration (10).
4. Amiodarone induced hepatotoxicity
Amiodarone represents a class III antiarrhythmic medication used to treat and prevent various
types of arrhythmias. Amiodarone can have serious side effects, including hepatotoxic effect. More
precisely this drug can cause DIFLD (drug-induced fatty liver disease). Therefore, application of
amiodarone is mainly recommended only for serious ventricular arrhythmias. Better understanding
of amiodarone induced liver injury molecular mechanism is necessary. One of the ways of reaching
that goal is to assess the effect of amiodarone in hepatic cell cultures. First step includes incubation
of hepatic cell cultures with different concentrations of amiodarone (e.g. 10 µM and 20 µM of
amiodarone hydrochloride 98%, diluted in 99% MetOH) for 24h and 48h. Afterwards, different
tests can be done. The Trypan blue dye exclusion test can be performed to determine concentration
of viable cells. Oil-Red-O stain is used for demonstrating triglyceride accumulation in the
hepatocytes. Following incubation with amiodarone, cells change their normal shape to oval, larger
shape, with increased accumulation and size of lipid droplets into the cytosol of amiodarone-

7. treated cells compared to controls. For determining the exact toxic concentrations of amiodarone
and their effect on cell viability, MTT assay can be used. Western blot can be performed by using
antibodies against adipophilin and PPARγ.
5. Tamoxifen induced hepatotoxicity
Tamoxifen is a selective oestrogen receptor modulator commonly used in therapy of breast cancer,
particularly in molecular subtypes that express oestrogen receptors (11). Nevertheless, long-term
use of tamoxifen is associated with various complications, including drug induced fatty liver
disease. Accordingly, prevention and treatment of DIFLD in breast cancer patients require the
elucidation of the molecular mechanism of tamoxifen-induced DIFLD. Studies on hepatic cell
lines can play major role in elucidating this pathological mechanism. Same as for amiodarone, first
step includes incubation of hepatic cell cultures with different concentrations of tamoxifen (e.g. 5
µM and 10 µM of tamoxifen hydrochloride 98%, diluted in 99% MetOH) for 24h and 48h.
Afterwards, different tests can be done. The Trypan blue dye exclusion test can be performed to
determine concentration of viable cells. Tamoxifen decreases cells viability stronger then
amiodarone. Following Oil-Red-O staining of cells incubated with tamoxifen, change of their
normal shape to oval, larger shape, macrovesicular steatosis and phospholipidosis is visualized.
For determining the exact toxic concentrations of amiodarone and their effect on cell viability,
MTT assay can be used. When performing RT-PCR, expression of SREBP-1c, PPARγ and
C/EBPα genes has been changed (through various studies). Western blot analyses should include
antibodies for some of these proteins: SREBP-1c, FAS, C/EBPα, PPARγ and MTP (12).
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10. Rivera P, Pastor A, Arrabal S, Decara J, Vargas A, Sánchez-Marín L, et al. Acetaminophen-Induced
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hepatocyte steatosis in vitro. Int J Mol Sci. 2014;15(3):4019-30.