screening for mechanisms of hepatotoxicity ...€¦ · screening for mechanisms of hepatotoxicity:...
TRANSCRIPT
SCREENING FOR MECHANISMS OF HEPATOTOXICITY: PHOSPHOLIPIDOSIS, STEATOSIS, APOPTOSIS AND INFLAMMATORY MARKERS
K.F. Marcoe, R. Keyser, P. TB. Nguyen, Y. Ovechkina, and C. O’DayMDS Pharma Services – Bothell, WA, USA
INTRODUCTIONDrug-induced hepatotoxicity is difficult to predict and remains a major cause for failures during drug development. Predictive toxicology screening assays for identifying biomakers and providing mechanistic assessments allow earlier identification of potential liabilities and can result in recommendations to minimize these risks during lead optimization as well as understand their relevance in provoking clinical hepatotoxicity. Development of an effective in vitro cell-based screening model to assess human hepatotoxicity potential of drugs requires the use of multiplexed technologies that utilize human hepatocytes and measure parameters at the single cell level, morphological and biochemical, investigative of pre-lethal cytotoxic effects, representative of different mechanisms of toxicity and suitable for rapid throughput. In this study we used multiplexed high content screening (HCS) with automated fluorescence microscopy and image analysis based technology (GE Healthcare INCell Analyzer 1000) to develop mechanistic cellular assays for assessment of hepatotoxicity in HepG2 cells and multiplexed sandwich immunoassays based on flowmetric Luminex xMap™ technology to measure inflammatory markers in these same cells. These in vitro cell-based assays provide a robust and rapid screening system for testing compound effects on cell proliferation, apoptosis, cell cycle, steatosis, phospholipidosis and cytokine secretion and other inflammatory markers. This cost-effective extensive multiplexed platform for predictive assessments delivers a more sensitive approach to detection of end-point-specific drug hepatotoxicities. Use of primary hepatocytes as the cell source for these assays is in development.
METHODSCell Culture Human hepatocellular carcinoma cell line (HepG2) was grown in MEM, 10% FBS, 1% NEAA, 1% Alanyl-L-Glutamine and 1% sodium pyruvate in tissue culture flasks, 75 mm2 and 225 mm2, in a humidified atmosphere of 5% CO2 at 37oC. Working stocks of log-phase HepG2 cells were cryo-preserved using a standard slow cooling in 10% DMSO protocol. Cells were thawed from working stocks and either passage once prior to seeding into plates or directly seeded into plates from cryo-preserved stocks.
Multiplexed Hepatotoxicity Assay Caspase-3 activation, a marker of apoptosis, phospho-histone-3, a marker of mitosis, and nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at 2x103 cells per well with complete growth media into 384-well Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5% CO2 at 37oC . Test compounds were serially diluted 3-fold over 10 concentrations and added 24 hours post cell seeding with a final assay concentration of 0.5% DMSO. Following an additional 72 hour incubation in the humidified atmosphere of 5% CO2 at 37oC, cells were fixed and immunolabeled with anti-active caspase-3 for detection of apoptosis and anti-phospho-histone-3 for detection of cell cycle and stained with a nuclei dye for cell proliferation quantification. Automated fluorescence microscopy was carried out using a GE Healthcare INCell Analyzer 1000, and images were collected with a 4X objective.
Multiplexed Hepato-Lipid Accumulation Assay Intracellular phospholipids, a marker of phospholipidosis, intracellular neutral lipid, a marker of steatosis, and nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at 4x103 cells per well with complete growth media into 384-well Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5% CO2 at 37oC for 24 hours. For detection of phospholipid accumulation a fluorescently-labeled phospholipid (Invitrogen, H34350) was added to the cells with test compounds serially diluted 3-fold over 10 concentrations in a final assay concentration of 0.5% DMSO. Following an additional 48 hour incubation in a humidified atmosphere of 5% CO2 at 37oC, cells were fixed and stained with a neutral lipid dye (Invitrogen, H34476) for neutral lipid detection and a nuclei dye for cell proliferation quantification. Automated fluorescence microscopy was carried out using a GE Healthcare INCell Analyzer 1000, and images were collected with a 4X objective.
Multiplexed Hepato-Cytokine Secretion Assay Cytokine secretion, as markers of inflammation, and nuclear count, as an index of cell proliferation, were measured in HepG2 cells. HepG2 cells were seeded at 8x103 cells per well with complete growth media into 96-well Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5% CO2 at 37oC for 24 hours. Cells were treated with LPS (30 µg/ml), TNFα (90 ng/ml), IL-1β (90 ng/ml) and acetaminophen (1 mM) serially diluted 3-fold over 8 concentrations. Final assay concentration of DMSO for acetaminophen was 0.05%. Following an additional 48 hour incubation in a humidified atmosphere of 5% CO2 at 37oC, supernatants were collected and cytokine detection was carried out using multiplexed sandwich immunoassays based on Luminex xMAP™ technology. To quantify cell proliferation, the monolayer of HepG2 cells remaining in each plate was immediately stained with Hoechst nuclear dye for normalization, and incubated 15 min at 37°C. Images were collected using a GE Healthcare INCell Analyzer 1000 with a 10X objective.
Data Analysis For HCS 12 bit tiff images were acquired using the INCell Analyzer 1000 3.2 and analyzed with Developer Toolbox 1.6 software. EC50 and IC50 values were calculated using nonlinear regression to fit data to a sigmoidal 4 point, 4 parameter One-Site dose response model, where: y (fit) = A + [(B – A)/(1 + (C/x) ^ D))]. Cytokine and inducer concentrations were interpolated from standard curves obtained by 5 point parameter sigmoidal nonlinear regression analysis. Curve-fitting and EC50 and IC50 calculations were performed using XLFit™ software (IDBS). Curves were generated with GraphPad Prism™ 3 software using a sigmoidal dose-response, variable slope model.
Measured Parameters Cell proliferation was measured by the signal intensity of the incorporated nuclear dye. The cell proliferation output was referred to as the relative cell count. To determine the cell proliferation end point, the cell proliferation data output was transformed to percent of control (POC) using the following formula:
POC = relative cell count (compound wells) x100 relative cell count (vehicle wells)
Relative cell count IC50 is the test compound concentration that produces 50% of the cell proliferation inhibitory response or 50% cytotoxicity level. A relative cell count EC50 is the test compound concentration that produces 50% of the maximum effective response that occurs at the curve inflection point. The output of each biomarker is fold increase over vehicle background normalized to the relative cell count in each well. Concentrations of test compound that cause a 5 or more fold induction in the caspase-3 signal were determined to induce significant apoptotic induction. When the fold induction of the phospho-histone-3 signal over background is ~1, there was “no effect” on the cell cycle. Two or more fold increase in phospho-histone-3 signal over vehicle background indicated significant test compound induction of mitotic block. Two or more fold decrease in the phospho-histone-3 signal may indicate G1/S block only when cytotoxicity levels are below the measured relative cell count IC80. When 2 or more fold decrease in the phospho-histone-3 signal are observed at concentrations higher than the relative cell count nuclear IC80, the decrease in mitotic cell counts are most likely due to a more general cytotoxicity effect rather than a true G1/S phase block. Wells with concentrations higher than the relative cell count nuclear IC80 are eliminated from the phospho-histone-3 analysis. Concentrations of test compound that cause a 5 or more fold induction of the labeled-phospholipid signal represent significant phospholipidosis induction. Concentrations of test compound which cause a 5 or more fold induction of the neutral lipid signal represent significant steatosis induction. Three or more fold increase in cytokine detection represents significant cytokine secretion.
MULTIPLEXED HEPATOTOXICITY ASSAY
CONCLUSIONIdentification of drug-induced hepatotoxic potential early in the drug development cascade can create opportunities for ranking and prioritizing, or developing alternatives with lower toxicity. We have developed a robust and rapid throughput screening system using HepG2 cells that allows early assessment of acute and chronic mechanisms of hepatotoxicity. Compounds with known hepatotoxicities were tested to validate the capabilities of this multiparametric HCS system in identifying and quantifying toxicities relevant to cell proliferation, apoptosis, cell cycle, steatosis, phospholipidosis. High concordance was found with the reported hepatotoxic profile for each compound tested. Further, we evaluated cytokine secretion in HepG2 cells to identify measurable biomarkers of inflammation. Significant secretion levels for 6 of the cytokines tested were confirmed thus validating this multiplexed approach for quantifying indications of hepatic inflammation. These hepatotoxicity screening assays are sensitive and reproducible and provide results that previously only have been attainable in more complex in vivo models. Our cost-effective in vitro multiplexed HCS platform offers comprehensive predictive information allowing pre-selection of drug scaffold designs with long-term hepatotoxicity considerations and may even have more relevance when performed in normal primary hepatocytes.
REFERENCES1. Dambach DM, Andrews BA, Moulin F. New technologies and screening srategies for hepatotoxicity: use on in vitro models. Toxicologic
Pathology, 2005;33:17-26. 2. Anderson N, Borlak J. Drug-induced phospholipidosis. FEBS Lett. 2006;580(23):5533-40.3. Nioi P, Perry BK, Wang E, et al. In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid-
based methodologies. Toxicology Sciences, 2007;99(1):162-173.4. Stonans I, Stonane E, RuBwurm S, et al. HepG2 human hepatoma cells express multiple cytokine genes. Cytokines, 1999;11(2):151-6.
The relative cell count IC50 (half maximal inhibitory constant) and EC50 (half maximal effective constant) values measure cell proliferation. Compound concentrations are indicative of 5-fold phospholipid and neutral lipid induction over vehicle background. All values are given as the mean ± s.e.m.
MULTIPLEXED HEPATO-CYTOKINE SECRETION ASSAY
A summary of detected cytokine secretion measured in HepG2 cells, (n =3). HepG2 cells were treated with LPS, TNFα, IL-1β and acetaminophen and screened for the secretory presence of 30 human inflammatory markers including IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, INFγ, INFα2a, IP-10, GM-CSF, G-CSF, MCP-1, MIP-1α, MIP-1β, TNFα, IL-1 receptor antagonist, Fibrinogen, CRP, Haptoglobin, SAA, Apo AI, Apo AII, Apo B, Apo CII, Apo CIII and Apo E.
A summary of Hepato-Lipid Accumulation Assay parameters for each compound tested, (n = 3).
31- 21- 11- 01- 9- 8- 7- 6-0
2
4
6
M ,]enitsalbniV[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
31- 21- 11- 01- 9- 8- 7- 6-0
02
04
06
08
001
M ,]enitsalbniV[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
31- 21- 11- 01- 9- 8- 7- 6-0
02
04
06
08
001
021
041
061
M ,]enitsalbniV[
Per
cen
t o
f C
on
tro
l
21- 11- 01- 9- 8- 7- 6- 5-0
02
04
06
08
001
021
041
061
M ,]eniropsoruatS[
Per
cen
t o
f C
on
tro
l
21- 11- 01- 9- 8- 7- 6- 5-0
02
04
06
08
001
M ,]eniropsoruatS[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
21- 11- 01- 9- 8- 7- 6- 5-0
1
2
3
4
5
6
M ,]eniropsoruatS[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
01- 9- 8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
041
061
M ,]A niropsolcyC[
Per
cen
t o
f C
on
tro
l
01- 9- 8- 7- 6- 5- 4- 3-0
02
04
06
08
001
M ,]A niropsolcyC[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
01- 9- 8- 7- 6- 5- 4- 3-0
2
4
6
M ,]A niropsolcyC[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
01- 9- 8- 7- 6- 5- 4- 3-0
1
2
3
4
5
6
M ,]lolonarporP[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
01- 9- 8- 7- 6- 5- 4- 3-0
02
04
06
08
001
M ,]lolonarporP[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
01- 9- 8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
041
061
M ,]lolonarporP[
Per
cen
t o
f C
on
tro
l
01- 9- 8- 7- 6- 5- 4- 3-0
2
4
6
M ,]nicymorhtyrE[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
01- 9- 8- 7- 6- 5- 4- 3-0
02
04
06
08
001
M ,]nicymorhtyrE[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
01- 9- 8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
041
061
M ,]nicymorhtyrE[
Per
cen
t o
f C
on
tro
l
sisotpopAnoitarefilorP lleC kcolB elcyC lleCnoitcudnI
Hepatotoxicity Assay, representative images are shown. Labels: Nuclei - green; Apoptotic cells - blue; Mitotic cells - red
Hepatotoxicity Assay, representative curves for cell proliferation, apoptosis induction, and cell cycle block are shown.
A summary of Hepatotoxicity Assay parameters for each compound tested, (n = 3).
The relative cell count IC50 (half maximal inhibitory constant) and EC50 (half maximal effective constant) values measure cell proliferation. Compound concentrations are indicative of a 5-fold apoptosis induction in activated caspase-3 signal and a 2-fold change in phospho-histone 3 signal (mitosis marker) over vehicle background. All values are given as the mean ± s.e.m.
Concentrations for cytokine induction and secretion are given as the mean ± s.e.m. Lipopolysaccharide (LPS), Tumor necrosis factor (TNF), Interleukin (IL), Interferon (INF), Interferon-gamma-inducible protein (IP), Granulocyte-macrophage-colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF), Monocyte chemoattractant protein (MCP ), Macrophage inflammatory protein (MIP), C-Reactive Protein (CRP), Serum Amyloid A (SAA), Apolipoprotein (Apo)
[LPS] µg/ml: wherecytokine secretion
3-fold over background_ _ _ _ _ _
Maximum [secreted cytokine], pg/ml
_ _ _ _ _ _
[TNFα] ng/ml: wherecytokine secretion
3-fold over background0.45 ± 0.08 0.29 ± 0.04 0.40 ± 0.05 0.110 ± 0.01 _ _
Maximum [secreted cytokine], pg/ml
10.64 ± 1.75 6454 ± 458 26.19 ± 2.53 87.83 ± 8.31 _ 5955 ± 29
[IL-1β] ng/ml: wherecytokine secretion
3-fold over background0.016 ± 0.003 < 0.04 _ 0.002 ± 0.000 0.81 ± 0.45 0.15 ± 0.05
Maximum [secreted cytokine], pg/ml
16.78 ± 0.78 5039 ± 39 _ 259 ± 12 6.64 ± 1.11 8.85 ± 1.29
[Acetam.] mM: wherecytokine secretion
3-fold over background_ _ _ _ _ _
Maximum [secreted cytokine], pg/ml
_ _ _ _ _ _(1 mM - 450 nM)
(30 - 0.01 µg/ml)
(90 - 0.04 ng/ml)
(90 - 0.04 ng/ml)
α
β
α α
dnuopmoC tnuoc llec evitaleR
CI 05)Morcim(
tnuoc llec evitaleRCE 05
)Morcim(
sisotpopA noitcudni
)Morcim(
sisotiM kcolb elcyc llec
)Morcim(
fo noitibihnI llec S/1G( sisotim
)kcolb elcyc)Morcim(
18.26lolonarporP ± 83.1632.5 ± 82.5583.5 ± 34.5 ——
630.0eniropsoruatS ± 720.0500.0 ± 412.0300.0 ± 910.0 — 441.0 ± 810.0
17.7AniropsolcyC ± 34.567.0 ± 99.903.0 ± 92.0 ——
200.0enitsalbniV ± 200.0000.0 ± 300.0000.0 ± 200.0100.0 ± 000.0 —
001 >001 >nicymorhtyrE ———
MULTIPLEXED HEPATO-LIPID ACCUMULATION ASSAYHepato-Phospholipid Accumulation Assay
Erythromycin
PropranololVehicle
Vehicle
VinblastineVehicle
Hepato-Phospholipid Accumulation Assay, representative images are shown. Labels: Nuclei - green; Phospholipids - red
8- 7- 6- 5- 4- 3-0
02
04
06
M ,]AniropsolcyC[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-002040608001021041061081002022042062
M ,]AniropsolcyC[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
M ,]AniropsolcyC[
Per
cen
t o
f C
on
tro
l
8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
M ,]nicymorhtyrE[
Per
cen
t o
f C
on
tro
l
8- 7- 6- 5- 4- 3-002040608001021041061081002022042062
M ,]nicymorhtyrE[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-0
01
02
03
04
05
06
M ,]nicymorhtyrE[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
M ,]lolonarporP[
Per
cen
t o
f C
on
tro
l
8- 7- 6- 5- 4- 3-002040608001021041061081002022042062
M ,]lolonarporP[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-0
01
02
03
04
05
06
M ,]lolonarporP[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
noitcudnI dipiL lartueNnoitcudnIdipilohpsohPnoitarefilorP lleC
noitcudnI dipiL lartueNnoitcudnIdipilohpsohPnoitarefilorP lleC
Hepato-Phospholipid Accumulation Assay, representative curves for cell proliferation and phospholipid and neutral lipid accumulation are shown.
Hepato-Neutral Lipid Accumulation Assay
Hepato-Neutral Lipid Accumulation Assay, representative images are shown. Labels: Nuclei - green; Neutral lipids - red.
Hepato-Neutral Lipid Accumulation Assay, representative curves for cell proliferation, phospholipid and neutral lipid accumulation are shown.
Cyclosporin AVehicle
8- 7- 6- 5- 4- 3-0
02
04
06
M ,]AniropsolcyC[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-002040608001021041061081002022042062
M ,]AniropsolcyC[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
M ,]AniropsolcyC[
Per
cen
t o
f C
on
tro
l
8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
M ,]nicymorhtyrE[
Per
cen
t o
f C
on
tro
l
8- 7- 6- 5- 4- 3-002040608001021041061081002022042062
M ,]nicymorhtyrE[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-0
01
02
03
04
05
06
M ,]nicymorhtyrE[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-0
02
04
06
08
001
021
M ,]lolonarporP[
Per
cen
t o
f C
on
tro
l
8- 7- 6- 5- 4- 3-002040608001021041061081002022042062
M ,]lolonarporP[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
8- 7- 6- 5- 4- 3-0
01
02
03
04
05
06
M ,]lolonarporP[
Fo
ld In
du
ctio
no
ver
Bac
kgro
un
d
noitcudnI dipiL lartueNnoitcudnIdipilohpsohPnoitarefilorP lleC
noitcudnI dipiL lartueNnoitcudnIdipilohpsohPnoitarefilorP lleC
— 01.53 ± 89.9001 >001 >nicymorhtyrE
04.9 ± 68.0 91.51 ± 01.1 37.8 ± 25.0 07.91 ± 66.5AniropsolcyC
34.59 ± 97.5 24.11 ± 31.1001 >001 >lolonarporP
noitcudni dipil lartueN)Morcim(
noitcudnidipilohpsohP)Morcim(
tnuoc llec evitaleRCE 05 )Morcim( ,
tnuoc llec evitaleRCI 05 )Morcim( ,
dnuopmoC