In Vitro Multiparameter Hepatotoxicity Assay Development in Human Primary Hepatocytes and HepG2 Cells with S9 Liver Fractions

1. IN VITRO MULTIPARAMETER HEPATOTOXICITY ASSAY DEVELOPMENT IN HUMAN PRIMARY
HEPATOCYTES AND HEPG2 CELLS WITH S9 LIVER FRACTIONS
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Marcoe, KF; Garside, HJ; Chesnut-Speelman, J; Foster, AJ; Ovechkina, Y; Warrior, U; Kenna, JG; Bowes, J and O’Day, C
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Ricerca Biosciences, Bothell, USA and AstraZeneca Safety Assessment UK, Innovative Medicines, Alderley Park, UK
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Introduction ROS AND MITOCHONDRIAL MEMBRANE POTENTIAL
ASSAY IN HUMAN PRIMARY HEPATOCYTES
ROS AND MITOCHONDRIAL MEMBRANE POTENTIAL
ASSAY IN HepG2 Cells
APOPTOSIS AND MITOSIS ASSAY IN HepG2 CELLS
WITH rS9, NADPH AND CYCLOPHOSPHAMIDE
Multiplexed apoptosis and cellular stress
in HepG2 cells
Drug-induced liver injury (DILI) in man is difficult to predict preclinically and a leading cause of drug attrition in clinical trials and
post approval withdrawals. Toxicology screening assays which utilize relevant cellular models and biomarkers directly linked to
mechanisms of cellular toxicity have the potential to enable early identification and deselection of compounds that have high Figure 1. Representative images of CCCP treatment effects on ROS Figure 3. Representative images of Nefazadone treatment effects on Figure 5. Representative curves for cell proliferation, apoptosis, and Figure 7. Representative images of geldanamycin treatment effects on
propensity to cause DILI. In this study, multiplexed high content screening (HCS) with automated fluorescence microscopy and generation and mitochondrial membrane potential in human primary ROS generation and mitochondrial membrane potential in HepG2 cell cycle after 24 hr cyclophosphamide exposure in HepG2 cells with apoptosis and cellular stress in HepG2 cells.
image analysis based technology was used to develop and evaluate cellular assays that detect key mechanisms considered
hepatocytes. cells. and without rS9 and NADPH in a 48 hour cytotoxicity assay, (n = 2).
relevant to DILI. Experiments were conducted in human primary hepatocytes and in HepG2 cells to explore time dependency of
the markers tested. Additionally, in some studies S9 liver fractions from Aroclor induced rats were added extracellularly to enhance Cytotoxic effects present only in HepG2 cells with rS9 and NADPH
the metabolic capability of HepG2 cells. A multiplexed format was used to evaluate the following parameters using the designated regeneration system.
reagents: cell proliferation/cell attachment (nuclear dyes); apoptosis (antibodies to activated caspase-3); cell cycle (antibodies to
phospho-Histone-H3); cellular stress (antibodies to HSP70/72); reactive oxygen species (ROS) generation (H2DFFDA staining), and
mitochondrial damage (TMRE staining). Our preliminary findings with a variety of hepatotoxic and non-hepatotoxic test compounds
demonstrate that this approach is a useful tool to evaluate the ability of test compounds to cause toxicity to isolated liver-derived
cells in vitro and indicate that further testing of a broad range of marketed drugs with and without DILI consequence in man is
warranted.
METHODS
Cell Culture Human hepatocellular carcinoma cell line (HepG2) was grown in RPMI1640, 10% FBS, 1% L-Alanyl-L-Glutamine
and 1% sodium pyruvate in a humidified atmosphere of 5% CO2 at 37oC. Cells were thawed from working stocks and passed
once prior to seeding into 384-well plates. Human primary hepatocytes (HPH, Invitrogen) from a single donor were thawed in Figure 8. Multiparameter Assay, represenative curves for cell proliferation,
hepatocyte culture medium with supplements and growth factors (HBM™, HCM™ SingleQuots® LONZA), treated with a percoll apoptosis, and cellular stress induction in HepG2 cells after 48 hour
separation step and immediately seeded into 384-well plates in the same media. Within 24 hours of plating these cells, the media
was exchanged with fresh media. Prior to compound addition, the media was exchanged again. For all assays test compounds
Geldanamycin exposure, (n = 2).
were serially diluted 3.16-fold over 10 concentrations with a final assay concentration of 0.5% DMSO. Compounds were added
Figure 2. Multiparameter Assay, representative curves for viable live Figure 4. Multiparameter Assay, representative curves for cell Figure 6. Multiparameter Assay, represenative curves for cell
24 or 48 hours post cell seeding in HPHs and 24 hours post seeding in HepG2 cells.
attached cells, ROS generation, and mitochondrial membrane potential proliferation, ROS generation , and mitochondrial membrane potential in proliferation, apoptosis, and cell cycle: a) 4 hr cyclophosphamide
in human primary hepatocytes after 4 and 24 hours of CCCP exposure HepG2 cells after 4 and 24 hours of Nefazadone exposure are shown. and rS9 + NADPH exposure to HepG2 cells in 48 assay, b) 24 hr
Multiplexed ROS Generation and Mitochondrial Membrane Potential Assay Intracellular staining with H2DFFDA (5-(and-6)- cyclophosphamide and rS9 + NADPH exposure to HepG2 cells in 48
are shown.
chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester, Invitrogen), a marker of ROS generation; TMRE (Invitrogen), a
marker of mitochondrial membrane potential induction; and Hoescht (Invitrogen) for nuclear count, an index of cell number, were
assay, c) 24 hr cyclophosphamide and rS9 + NADPH exposure to HepG2
measured after 4 and 24 hours of test compound incubations in HepG2 cells and HPHs. For optimal well cell coverage at each cells in 72 hr assay, (n = 2).
time point HepG2 cells were seeded in 384-well tissue culture plates at 4x103 and 3x103 cells per well, respectively, with complete
growth media. HPHs were seeded at 30x103 cells per well with hepatocyte culture medium with supplements and growth factors
into 384-well Collagen I coated optical plates (BD Biosciences). Plated cells were incubated in a humidified atmosphere of 5% CO2
at 37oC prior to and for the duration of test compound Incubation. Following 4 and 24 hour compound incubation periods, cells
were washed and incubated with the multiplexed dyes for 30 minutes.
Table 5. Cell proliferation, apoptosis and HSP70/72 parameters for
geldanamycin after 48 hour exposure to HepG2 cells, (n = 2). HSP70/72
Multiplexed Apoptosis and Mitosis Assay Caspase-3 activation, a marker of apoptosis, phospho-Histone-H3, a marker of
mitosis, and nuclear count, an index of cell proliferation, were measured in HepG2 cells without and with addition of S9 liver
signal more sensitive marker of early cell distress.
fractions from Aroclor induced rats (rS9, Molecular Toxicology) and NADPH regeneration system (BD Biosciences). HepG2 cells
were seeded at 2x103 cells per well with complete growth medium into 384-well tissue culture plates and incubated in a humidified
atmosphere of 5% CO2 at 37oC. Prior to test compound addition the medium was exchanged with complete growth medium or
Relative cell
medium containing rS9 with and without the NADPH regeneration system. Following compound addition, the medium in the cell count
Apoptosis
5X Fold
Max
Apoptosis
HSP70/72
5X Fold
Max
HSP70/72
Compound Cell line
plates was exchanged at 4 or 24 hours with fresh complete growth medium. For experiments with 4 hour compound/rS9 exposure, IC50 Induction Fold Induction Fold
(microM) (microM) Induction (microM) Induction
the treated cell plates were incubated a total of 48 hours prior to being fixed and immunolabeled with anti-active caspase-3 for
17-(Allylamino)-17-
detection of apoptosis and anti-phospho-Histone-H3 for detection of cell cycle and stained with the nuclei dye, DAPI, for cell demethoxygeldanamycin HepG2 0.036 ± 0.002 0.03 ± 0.002 122 ± 46 0.006 ± 0.0005 391 ± 120
proliferation quantification. For experiments with 24 hour compound/rS9 exposure, the treated cell plates were incubated a total The relative cell count IC50 (half maximal inhibitory constant) value measures cell proliferation. Compound concentrations
of 48 or 72 hours prior to fixing and staining. are indicative of a 5-fold induction in apoptosis signal and 5-fold induction in HSP70/72 signal over vehicle background.
All values are given as the mean ± s.e.m.
Multiplexed Apoptosis and Cellular Stress Assay Caspase-3 activation, a marker of apoptosis, HSP70/72, a marker of cellular
stress, and nuclear count, an index of cell proliferation, were measured in HepG2 cells. HepG2 cells were seeded at 2x103 cells
per well with complete growth medium into 384-well tissue culture plates and incubated in a humidified atmosphere of 5% CO2
at 37oC. Test compounds were added 24 hours post cell seeding. Following an additional 48 hour incubation, cells were fixed and
immunolabeled with anti-active caspase-3 for detection of apoptosis and anti-HSP70/72 (Enzo Life Sciences) for detection of
Table 2. A comparison of the percent of attached live cell count IC50,
H2DFFDA, and TMRE parameters for each compound after 4 and 24
Table 3. A comparison of the relative cell count IC50, H2DFFDA, and
TMRE parameters for each compound after 4 and 24 hours exposure
Table 4. A comparison of multiplexed data (cell proliferation, apoptosis,
and cell cycle) after 4 and 24 hour compound and rS9 + NADPH exposure Conclusions
cellular stress and stained with the nuclei dye, DAPI, for cell proliferation quantification. hours exposure to human primary hepatocytes (HPH), (n = 2). to HepG2 cells, (n = 2). to HepG2 cells in 48 and 72 hour cytotoxicity assay (n = 2). Transient
effect was observed on mitosis. Development of a comprehensive predictive toxicology testing platform requires
selection of relevant in vitro cellular models capable of drug metabolism and
Measured Parameters For HepG2 cells, proliferation was measured by the signal intensity of the incorporated nuclear dye. The Relative Apoptosis Max G2/M cell
G2/M cell mechanistic hepatic toxicity assessments.
% of attached H2DFFDA Max TMRE Relative cell H2DFFDA Max TMRE Compound /
Compound / cell count
Assay
Relative
5X Fold
Apoptosis
Apoptosis
Max
cycle
G1/S cell
G1/S cell The multiplexed HCS platform with
cell proliferation output was referred to as the relative cell count. To determine the cell proliferation end point, the cell proliferation Compound live cells
Compound
% of attached
3.5X Fold
H2DFFDA
H2DFFDA
Max
0.5X Fold
TMRE Compound
count
Compound
Relative cell
3.5X Fold
H2DFFDA
H2DFFDA
Max
0.5X Fold
TMRE Compound Cell line r S 9 + N A D P H Assay cell count 5X Fold Apoptosis cycle block
cycle
(microM) automated fluorescence microscopy and image analysis described here enables
Compound Cell line exposure live cells 3.5X Fold H2DFFDA 0.5X Fold Compound Cell line exposure count 3.5X Fold H2DFFDA 0.5X Fold Compound + NAD
Duration IC 50
Cell line r S 9 RelativeP H Apoptosis Max Fold
Induction G2/M cell block cycle block
Compound IC
Cell line % of attached 50 H2DFFDA
exposure Induction Max Fold TMRE Induction Compound cell IC50
Relative exposure H2DFFDA Induction Induction TMRE Fold
Cell line Fold Induction Compound / treatment Duration IC 50 Fold
Induction G1/S cell block
data output was transformed to percent of control (POC) using the formula shown below. For human primary hepatocytes attached Compound (hours) IC50 Induction Fold Induction Compound (hours) IC50 Max Induction Assay cell count
treatment (microM)
(microM) Apoptosis
5X Fold Induction
cycle (microM) (microM)
Compound Cell line exposure
(hours)
(microM)3.5X Fold
live cells (microM)
(microM) H2DFFDA Induction0.5X Fold
(microM)
(microM)
Induction (microM) Compound Cell line exposure
count (hours) 3.5X Fold (microM)
(microM)
(microM) H2DFFDA Induction Fold (microM)
(microM) 0.5X Induction (microM)
Compound Cell line r S 9 + N A D P H
Duration IC 50 Induction
(microM)
Fold
(microM) cycle block
block
Induction (microM)
detection of multiple key mechanisms considered relevant to DILI in human primary
live cells were measured by the signal defined by nuclear segmentation of the incorporated nuclear dye. The live cell output was IC50 Induction Fold Induction IC50 Induction Fold Induction CCCP treatment
HepG2 4hr 48hr 27 ± 5 45 ± 19 29 ± 6 (microM)
N/A 72 ± 0.1
(hours)
(microM) (microM) Induction (microM)
(hours)
(microM) (microM) Induction (microM) CCCP HepG2 (microM)
4hr (microM)
48hr Induction
27 ± 5 (microM)
45 ± 19 29 ± 6 N/A hepatocytes (HPH) as well as metabolically modified HepG2 cells. Drug induced
72 ± 0.1
referred to as the percent of attached live cells. To determine the live cell end point, the live cell data output was transformed to CCCP HPH 4 > 250 216 ± 35 4.9 ± 0.8 N/A CCCP HepG2 4 173 ± 7 167 ± 4 4.9 ± 0.2 2.7 ± 1.1 CCCP
CCCP
HepG2 24hr
HepG2
48hr
24hr
52 ± 1
48hr
24 ± 2
52 ± 1
43 ± 11
24 ± 2
N/A
43 ± 11
0.12 ± 0.03
N/A 0.12 ± 0.03
CCCP HPH 4 > 250 216 ± 35 4.9 ± 0.8 N/A CCCP HepG2 4 173 ± 7 167 ± 4 4.9 ± 0.2 2.7 ± 1.1 CCCP HepG2
CCCP
CCCP Chlorpromazine CCCP
4hr
HepG2 48hr
24hr
HepG2
27 ± 5
72hr
24hr 1
45 ±±19
42 10
72hr
44 ± 6
29 ± 19
42 ± 10
N/A± 33
42 72 ±N/A
0.1
44 ± 19 0.12 ± 0.03
42 ± 33
71
N/A ROS and decreased mitochondrial membrane potential were detectable in HPHs and
71
POC using the same formula: Chlorpromazine HPH HPH
CCCP 4 HPH 4 > 250 4 100 216 ± > 100 N/A 4.9 ± 0.81.2 ± 0.4
> 35 N/A ± 0.4 ± 22
60.7 CCCP ChlorpromazineHepG2 HepG2 4 4
HepG2 173 ± 7 4
51 ± 2 167 ± 4 29 ± 4.9 ± 0.2 9.9 ± 0.8 ± 1.1
4 2.7 9.9 ± 0.8 N/A HepG2 24hr
HepG2 48hr
4hr 52 ±
48hr 24 ±±26
54 43N/A
± 11 N/A 0.4
2± N/A N/A
5 ± 2 HepG2 cells with generation of ROS more pronounced at 4 hours than 24 hours for
Chlorpromazine N/A 1.2 60.7 ± 22 Chlorpromazine 51 ± 2 29 ± 4 N/A Chlorpromazine HepG2 4hr 10
CCCP Chlorpromazine
HepG2 24hr
HepG2 72hr
24hr 42 48hr
± 4448hr12
± 19