1. SCREENING FOR MECHANISMS OF HEPATOTOXICITY: PHOSPHOLIPIDOSIS, STEATOSIS,
APOPTOSIS AND INFLAMMATORY MARKERS
K.F. Marcoe, R. Keyser, P. TB. Nguyen, Y. Ovechkina, and C. O’Day
MDS Pharma Services – Bothell, WA, USA
INTRODUCTION MULTIPLEXED HEPATOTOXICITY ASSAY MULTIPLEXED HEPATO-LIPID ACCUMULATION ASSAY
Drug-induced hepatotoxicity is difficult to predict and remains a major cause for failures during drug development. Predictive toxicology Hepato-Phospholipid Accumulation Assay A summary of Hepato-Lipid Accumulation Assay parameters for each compound tested, (n = 3).
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 Relative cell count Relative cell count Phospholipid induction Neutral lipid induction
hepatotoxicity. Development of an effective in vitro cell-based screening model to assess human hepatotoxicity potential of drugs requires Compound IC50, (microM) EC50, (microM) (microM) (microM)
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. Propranolol > 100 > 100 11.42 ± 1.13 95.43 ± 5.79
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
Cyclosporin A 19.70 ± 5.66 8.73 ± 0.52 15.19 ± 1.10 9.40 ± 0.86
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 Vehicle Vinblastine
cycle, steatosis, phospholipidosis and cytokine secretion and other inflammatory markers. This cost-effective extensive multiplexed platform Vehicle Propranolol Erythromycin > 100 > 100 35.10 ± 9.98 —
for predictive assessments delivers a more sensitive approach to detection of end-point-specific drug hepatotoxicities. Use of primary Hepatotoxicity Assay, representative images are shown. Labels: Nuclei - green; Apoptotic cells - blue;
hepatocytes as the cell source for these assays is in development. Mitotic cells - red 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.
Ce l l Pr ol i f e r at i on Apopt osi s I n d u c t i o n Ce l l C y c l e B l o c k
METHODS
MULTIPLEXED HEPATO-CYTOKINE SECRETION ASSAY
160 100 6
Cell Culture Human hepatocellular carcinoma cell line (HepG2) was grown in MEM, 10% FBS, 1% NEAA, 1% Alanyl-L-Glutamine and 1%
Percent of Control
over Background
over Background
140
Fold Induction
Fold Induction
80
120
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 100
80
60
4
HepG2 cells were cryo-preserved using a standard slow cooling in 10% DMSO protocol. Cells were thawed from working stocks and either 60 40
2 A summary of detected cytokine secretion measured in HepG2 cells, (n =3). HepG2 cells were treated with LPS, TNFα, IL-1β and acetaminophen
40
passage once prior to seeding into plates or directly seeded into plates from cryo-preserved stocks. 20
20
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-
0
-10 -9 -8 -7 -6 -5 -4 -3
0
-10 -9 -8 -7 -6 -5 -4 -3
0
-10 -9 -8 -7 -6 -5 -4 -3 Vehicle Erythromycin 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,
Multiplexed Hepatotoxicity Assay Caspase-3 activation, a marker of apoptosis, phospho-histone-3, a marker of mitosis, and [Cyclosporin A], M [Cyclosporin A], M [Cyclosporin A], M
Apo AI, Apo AII, Apo B, Apo CII, Apo CIII and Apo E.
nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at 2x103 cells per well with complete 160 100 6 Hepato-Phospholipid Accumulation Assay, representative images are shown. Labels: Nuclei - green;
growth media into 384-well Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5%
Percent of Control
over Background
over Background
Phospholipids - red
140
Fold Induction
Fold Induction
5
80
120
CO2 at 37oC . Test compounds were serially diluted 3-fold over 10 concentrations and added 24 hours post cell seeding with 100 60
4
α α
a final assay concentration of 0.5% DMSO. Following an additional 72 hour incubation in the humidified atmosphere of 5%
80 3
60 40
2 Cell Proliferation Phospholipid Induction Neutral Lipid Induction [LPS] µg/ml: where
CO2 at 37oC, cells were fixed and immunolabeled with anti-active caspase-3 for detection of apoptosis and anti-phospho- 40
20
20
1 Cell Proliferation Phospholipid Induction Neutral Lipid Induction cytokine secretion _ _ _ _ _ _
histone-3 for detection of cell cycle and stained with a nuclei dye for cell proliferation quantification. Automated fluorescence 3-fold over background
120 260 60
0 0 0 240
(30 - 0.01 µg/ml)
Percent of Control
over Background
over Background
-10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 120
100 260
220 60
50
Maximum [secreted cytokine],
Fold Induction
Fold Induction
240
microscopy was carried out using a GE Healthcare INCell Analyzer 1000, and images were collected with a 4X objective.
200
_ _ _ _ _ _
Percent of Control
[Propranolol], M [Propranolol], M [Propranolol], M
over Background
over Background
180
00 220
180 50
Fold Induction
Fold Induction
40
80
60
200
160
1 80
140 40
30
pg/ml
[TNFα] ng/ml: where
160
120
60 140
100
40 30
20
Multiplexed Hepato-Lipid Accumulation Assay Intracellular phospholipids, a marker of phospholipidosis, intracellular neutral
180
20
cytokine secretion 0.45 ± 0.08 0.29 ± 0.04 0.40 ± 0.05 0.110 ± 0.01 _ _
α
160 10 0 6 160
00
40
20 20
10
80
40
Percent of Control
over Background
over Background
140
lipid, a marker of steatosis, and nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at
60
5 20
3-fold over background
Fold Induction
Fold Induction
80 20
0 10
(90 - 0.04 ng/ml)
120 40
0 0
4 -8 -7 -6 -5 -4 -3 20 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3
100 0 0 0
Maximum [secreted cytokine],
4x103 cells per well with complete growth media into 384-well Collagen I coated optical plates (BD Sciences) and incubated in
60
80 3 -8 -7 [Pro6 ranolol], M
-p -5 -4 -3 -8 -7 [Pro6 ranolol], M
-p -5 -4 -3 -8 -7 [Pro6 ranolol], M
-p -5 -4 -3
10.64 ± 1.75 6454 ± 458 26.19 ± 2.53 87.83 ± 8.31 _ 5955 ± 29
60 40 [Propranolol], M [Propranolol], M [Propranolol], M pg/ml
a humidified atmosphere of 5% CO2 at 37oC for 24 hours. For detection of phospholipid accumulation a fluorescently-labeled
2
[IL-1β] ng/ml: where
40
20
1
20
phospholipid (Invitrogen, H34350) was added to the cells with test compounds serially diluted 3-fold over 10 concentrations cytokine secretion 0.016 ± 0.003 < 0.04 _ 0.002 ± 0.000 0.81 ± 0.45 0.15 ± 0.05
β
0 0 0
-12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5
120 260 60
240
3-fold over background
Percent of Control
in a final assay concentration of 0.5% DMSO. Following an additional 48 hour incubation in a humidified atmosphere of 5%
over Background
over Background
[Staurosporine], M [Staurosporine], M [Staurosporine], M 120
100 260
220 60
50
Fold Induction
Fold Induction
240
200
(90 - 0.04 ng/ml)
Percent of Control
over Background
over Background
00
180 220
180 50
Maximum [secreted cytokine],
Fold Induction
Fold Induction
40
CO2 at 37oC, cells were fixed and stained with a neutral lipid dye (Invitrogen, H34476) for neutral lipid detection and a nuclei 16.78 ± 0.78 5039 ± 39 _ 259 ± 12 6.64 ± 1.11 8.85 ± 1.29
200
160