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Title Page

Long term chronic toxicity testing using human pluripotent stem cell-derived hepatocytes.

Gustav Holmgren, Anna-Karin Sjögren, Isabel Barragan, Alan Sabirsh, Peter Sartipy, Jane Synnergren, Petter

Björquist, Magnus Ingelman-Sundberg, Tommy B. Andersson, Josefina Edsbagge

Affiliation:

Systems Biology Research Center, School of Bioscience, University of Skövde, Sweden

(G.H., J.S., P.S.)

Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of

Gothenburg, Sweden

(G.H.)

Cellectis AB, Gothenburg, Sweden

(P.S., J.E., P.B.*)

*Current address: NovaHep AB, Gothenburg, Sweden

Department of Drug Metabolism and Pharmacokinetics, AstraZeneca R&D Mölndal, Sweden

(T.A.)

Department of Discovery Safety, Drug Safety and Metabolism, AstraZeneca R&D Mölndal, Sweden

(A-K.S.)

Department of Bioscience, Cardiovascular and Metabolic Diseases, AstraZeneca R&D Mölndal, Sweden

(A.S.)

Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm,

Sweden

(I.B., T.A., M.I-S.)

DMD Fast Forward. Published on June 30, 2014 as doi:10.1124/dmd.114.059154

Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.

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Running Title Page

Long term toxicity testing using hPSC-derived hepatocytes

Corresponding Author:

Gustav Holmgren,

Systems Biology Research Centre, School of Bioscience, University of Skövde, Sweden

Postal Address: Visiting address:

Box 408 Kanikegränd 3A

541 28 Skövde, Sweden

Mobile: +46 703 300 335

Fax: +46 500 416325

Email: Gustav.holmgren@his.se

Text pages: 22

Number of tables: 1

Number of figures: 3

Number of references: 38

Abstract: 177 words

Introduction: 727 words

Discussion: 969 words

Abbreviations:

ALT, alanine aminotransferase; CYP, cytochrome P450; DILI, drug induced liver injury; DMSO, dimethyl

sulfoxide; FBS, fetal bovine serum; HCA, high content analysis; hESC, human embryonic stem cells; hiPSC,

human induced pluripotent stem cells; hiPS-HEP, human induced pluripotent stem cell-derived hepatocytes;

HNF4a, hepatocyte nuclear factor 4 alpha; PHH, primary human hepatocytes; hPSC, human pluripotent stem

cells; ROS, reactive oxygen species

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Abstract

Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new

models for in vitro drug testing of e.g. toxicity and pharmacological effects. Late stage attrition in the

pharmaceutical industry is to a large extent caused by selection of drug candidates using non-predictive pre-

clinical models which are not clinically relevant. The current hepatic in vivo and in vitro models show clear

limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of

using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were

incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived

hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to

conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to

the best of our knowledge, the first report of a long term toxicity study using hPSC-derived hepatocytes, and the

observations support further development and validation of hPSC-based toxicity models for evaluating novel

drugs, chemicals and cosmetics.

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Introduction

The liver is one of the most affected organs involved in drug related toxicity. More than 30 % of the terminations

of drug development programs are caused by adverse effects in the liver, and liver toxicity represents almost 20

% of the safety-related, post-market withdrawals (Ballet 1997).

The pharmaceutical industry currently lacks relevant in vitro models that can predict human liver toxicity early

in the drug development process. The gold standard of in vitro models for hepatotoxicity is primary human

hepatocyte (PHH) cultures, which is considered the most relevant model for human liver toxicology applications

currently available (Guillouzo and Guguen-Guillouzo 2008). However, the low availability, large donor

variation, and the rapid loss of metabolic activity following in vitro culture limits their usefulness, in particular

for detecting chronic toxicity (Guguen-Guillouzo and Guillouzo 2010). One alternative to the PHH that has

emerged during recent years is the human hepatocellular carcinoma cell line HepaRG, a bi-potent progenitor cell

line with the ability to differentiate into hepatocyte- and biliary-lineages (Gripon et al. 2002). The differentiated

hepatocyte-like cells retain many of the functions of PHH, and the potential to use HepaRG in metabolism, and

toxicity studies has been reported (Andersson et al. 2012; Aninat et al. 2006; Antherieu et al. 2010; Kanebratt

and Andersson 2008) However, further evaluation of HepaRG as a toxicity model is needed, since a direct

comparison between HepaRG, PHH and HepG2 indicated a low sensitivity for detection of hepatotoxic drugs in

HepaRG (Gerets et al. 2012). Another extensively used hepatic cell model is the HepG2 tumor cell line (Sassa et

al. 1987). Despite the frequent usage of these cells, the resemblance to PHH is low and the fact that these cells

proliferate in culture make them impractical for long term studies (Gerets et al. 2009; Gerets et al. 2012; Xu et al.

2004).

Other novel in vitro models have been evaluated for their usefulness in studies of long term drug exposure.

Micro-patterned co-cultures of PHH and murine stromal fibroblasts displayed a functional stability to enable a 9

days repeated-dose exposure with 65.7 % sensitivity to detect drug induced liver injury (DILI) (Khetani et al.

2013). There are also reports of a canine co-culture model of hepatocytes and non-parenchymal stromal cells

with metabolic capacities maintained for up to 2 weeks and a superiority for the classification of DILI

compounds compared to HepG2 and PHH during 5 day compound exposure (Atienzar et al. 2014). However,

prediction of liver toxicity using different cell models needs further investigations since sensitivity and

specificity varies, a result which could be due to time of exposure, selection of investigated substances and the

specific cell type used (Gerets et al. 2012).

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Human pluripotent stem cells (hPSC) offer a new approach for generating cells for in vitro models. The ability of

unlimited propagation and the potential to differentiate into all cell types in the human body makes hPSC very

attractive (Takahashi et al. 2007; Thomson et al. 1998). Protocols to differentiate human embryonic stem cells

(hESC) and human induced pluripotent stem cells (hiPSC) into hepatocytes have improved immensely in recent

years, and now hepatocyte cultures with high purity and many characteristics of functional hepatocytes can be

obtained from hPSC (Brolen et al. 2010; Si-Tayeb et al. 2010; Szkolnicka et al. 2014; Touboul et al. 2010;

Ulvestad et al. 2013; Yildirimman et al. 2011; Hannan et al. 2013). The fact that these cells can be derived in a

robust and reproducible fashion from a well characterized source makes them very attractive as a toxicity

assessment model.

In this study, we have evaluated the potential of using hiPSC-derived hepatocytes to study hepatotoxicity in

response to chronic drug exposure. Earlier studies have shown that in many cases repeated dosing and exposure

for 2-weeks is necessary to be able to detect human toxicity of drugs (Olson et al. 2000; Roberts et al. 2013). The

hepatocytes used in our study, hiPS-HEP, were exposed to relevant concentrations of hepatotoxic compounds for

2, 7, and 14 days of repeated dosing. The cell morphology was monitored during the entire time period and the

cell viability, as well as the induction of steatosis and phospholipidosis, was assessed at each time point.

Importantly, the data show that the hiPS-HEP model is stable enough to enable at least a two-week study of drug

exposure, and that it is specific enough to assess hepatotoxic responses on a mechanistic level.

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Materials and Methods

Cell culture

Human induced pluripotent stem cell-derived hepatocytes (hiPS-HEPTM) were obtained from Cellectis AB

(www.cellectis.com) and cultured according to the instructions from the manufacturer. The cells were

maintained in 96-well plates and medium was replaced every second day during the compound incubation. The

human hepatocellular carcinoma cell line HepG2 was used as a control cell model. The HepG2 cells were

purchased from ATCC (HB-8065, lot#59635738) and cultured in Dulbecco's Modified Eagle Medium (DMEM,

high glucose, GlutaMAX™ Supplement, pyruvate, 31966-021, Gibco/Life Technologies) supplemented with 10

% heat inactivated FBS (16140-071, Gibco/Life Technologies), 1 % penicillin-streptomycin (Pen Strep, 15140-

035, Gibco/Life Technologies) and 1 % non-essential amino acids (MEM NEAA, 11140-035, Gibco/Life

Technologies).

Materials

Four model compounds known to cause hepatotoxicity were used in this study. Amiodarone (A8423), aflatoxin

B1 (A6636), and troglitazone (T2573) were purchased from Sigma Aldrich (Sweden). Ximelagatran was kindly

provided by AstraZeneca, R&D (Mölndal, Sweden). All compounds were purchased as powder and dissolved in

DMSO (D2650-100ml) to a stock solution concentration 250x higher than the top assay concentration (see Table

1 for details on compounds). EZ4U Cell proliferation and Cytotoxicity assay was purchased from

Biomedica/Biotech-IgG. The fluorescent probes for the imaging analysis, Hoechst 33342, HCS LipidTOX green

neutral lipids (H34475) and HCS LipidTOX red phospholipidosis (H34351) were acquired from Life

technologies, Invitrogen.

Dose-response study and viability testing

The hiPS-HEP were exposed to various concentrations of the compounds for three different time points; one

single-dose exposure for 2 days and repeated dose exposures for 7- (three repeated doses) and 14 days (6

repeated doses), respectively. Each compound was half-log diluted in DMSO from a DMSO stock solution and

then further diluted in a two-step procedure, first 25 x dilutions in cell culture medium followed by a 10 x

dilution in the cell culture plates. The final concentration of DMSO was 0.4 %. Every sample was assayed in

triplicate wells for each time point. After incubation with the compounds, the cell viability was assessed using

EZ4U cell proliferation and cytotoxicity assay according to the instructions from the provider. The absorbance at

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492 nm was measured in a FLUOstar Omega plate-reader (BMG LABTECH) and the samples were blank

corrected and normalized to vehicle control.

Dose-response curves were plotted as percentage of vehicle control using Graphpad Prism 4 and EC10 values

were generated using non-linear sigmoidal dose-response regression. Since the values were normalized to the

vehicle control, constraints of TOP= 100 and BOTTOM=0 were included in the regression setup.

Assessment of steatosis and phospholipidosis

To assess the induction of steatosis and phospholipidosis following compound exposure, cells stained with

fluorescent probes for neutral lipids and phospholipids were measured using quantitative image analysis. To

avoid non-specific general cytotoxic effects, the concentration used for each compound was the EC10 from the

14 day dose-response study. The following concentrations were used; amiodarone: 2.5 µM; aflatoxin B1: 0.1

µM; troglitazone: 40 µM; ximelagatran: 10 µM. At the last medium change, 48 hours before the termination of

the experiment, the HCS LipidTOX Red Phospholipidosis substrate was added to the cells together with the

compounds. At termination of the study the cell viability was assessed, using the EZ4U kit as described above,

and the cells were fixed in 4 % formaldehyde for 30 minutes. After subsequent washing steps with DPBS +/+,

Hoechst stain, to a final concentration of 10µg/ml (1000-fold dilution), was added to the wells and the cells

incubated at room temperature for 20 minutes. Alternatively, to assess the steatosis, the HCS LipidTOX Green

Neutral Lipid was diluted 1000-fold in DPBS+/+ and added to the cells. The cells were kept at room temperature

for a minimum of 30 minutes prior to storage at +4°C until further analysis.

Automated Imaging and Analysis

Cell cultures in 96-well imaging plates (BD Falcon) were imaged using an automated robotic microscope

(ImageXpress Micro, from Molecular Devices) fitted with a 10x Nikon plan fluor objective (NA 0.3). Image

acquisition and processing was performed using MetaXpress software (version 5.1, from Molecular Devices). To

accommodate the variable thickness of these cultures and to facilitate image analysis, focal stacks were taken for

each wavelength and processed to produce single images containing only in-focus pixels. Cellular nuclei were

then identified in each image and classified as positive for steatosis and phospholipidosis based on multiple

thresholds for lipid morphology, staining intensity, and the area stained for each cell. Data are reported as

percentage of positive cells where the total population was defined by the nuclear staining.

Statistical analysis

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To test whether the dose-response curves significantly differed at each time point an F-test was performed using

GraphPad Prism 4. A p-value ≤ 0.05 was considered statistically significant.

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Results

hiPS-HEP characteristics

The drug metabolism and transporter functions in the hiPS-HEP have previously been characterized in detail,

and compared to cryopreserved PHH cultured for 4 and 48 hours (Ulvestad et al. 2013). Notably, the hiPS-HEP

show stable gene- and protein expression as well as functional activity of transporters and CYPs during in vitro

culture. In the present investigation we focused on prediction of hepatotoxicity using hiPS-HEP in long term

cultures. At the start of the experiments, the hiPS-HEP formed a highly homogeneous (>90 %) monolayer of

hepatocytes with a morphology resembling PHH. The cells displayed typical polygonal shape, clear cell-cell

borders, and prominent light nuclei surrounded by dark cytoplasm (Figure 1 A). The cells expressed several

hepatocyte-associated markers, such as cytokeratin 18 and HNF4α, and showed ability to store glycogen, as

illustrated in figure 1 B – D. Notably, the hiPS-HEP exhibited some cytochrome P450 enzyme activity at levels

comparable to that of PHH cultured for 48 hours. Specifically, the hiPS-HEP showed ~30 % of CYP1A and

more than 4-fold higher CYP3A activity (Ulvestad et al. 2013). The cells expressed these characteristics stably

for more than two weeks, without any sign of proliferation, which allowed for the set-up of an extensive repeated

dose testing.

hiPS-HEP sensitivity to hepatotoxic compounds

As shown in Figure 2, the initial dose-response study demonstrated that the hiPS-HEP were sensitive to several

of the compounds tested, indicated by the drop in viability at increased concentration of amiodarone, aflatoxin

B1, and troglitazone. For troglitazone, the viability dropped from approximately 100 % to near 0 % within a

narrow concentration range. The hiPS-HEP responded strongly to amiodarone and aflatoxin B1 in a time-

dependent manner. A significant (p<0.001) shift of the viability curve was observed for 7 (Figure 2, blue) and 14

days exposure (Figure 2, black) compared to the 2 days exposure (Figure 2, red). This indicates that the cells are

more sensitive over long-term, repeated dose exposures compared to short-term single dose exposures. The

hiPS-HEP were non-responsive to ximelagatran. Comparable levels of sensitivity were observed for HepG2 and

hiPS-HEP cells when exposed to the compounds for 48 hours (Figure 2, green), whereas it was not possible to

use HepG2 for the long term treatment due to the high extent of cell proliferation.

Induction of steatosis and phospholipidosis

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In order to evaluate if the hiPS-HEP responded to the compounds in a hepatocyte specific way, we assessed the

compounds´ ability to induce steatosis and phospholipidosis. For these experiments, a sub-cytotoxic

concentration (EC10 at day 14) of each compound was selected. As shown in Figure 3 A, the cell viability at

each time point was high (around 90%). Amiodarone induced phopholipidosis in hiPS-HEP (Figure 3 B) after 7

and 14 days exposure. Representative images of the amiodarone-induced phospholipidosis, in comparison to the

control, are shown in Figure 3 D. Steatosis was detected in cells dosed with troglitazone (Figure 3 C) for 2 days,

and with increasing number of positively stained cells after 7 and 14 days exposure.

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Discussion

There is a great need in the pharmaceutical industry today for more predictive cell-based models for toxicity

assessment. In this study, we demonstrate the potential of using hPSC-derived hepatocytes as an in vitro model

for long-term hepatotoxicity. The hiPS-HEP represent a robust and reproducible population of cells with many

hepatic characteristics that can be maintained for at least two weeks in culture, thus enabling a repeated-dose

chronic exposure to hepatotoxic compounds. The hepatocytes respond to the compounds in a dose-dependent

manner and show increasing sensitivity to amiodarone and aflatoxin B1 during long term exposure. The

induction of steatosis and phospholidiposis can also be assessed in order to evaluate the hepatocyte-specific toxic

response after compound exposure. The hiPS-HEP display a clear phospholipidosis when exposed to

amiodarone, and the effect increases during the exposure time period. Troglitazone induces steatosis in the cells

in a similar manner, with increasing number of positive cells with time.

The expression and activity of several important metabolizing CYP enzymes and transporter proteins in hiPS-

HEP have previously been investigated (Ulvestad et al. 2013). Those studies revealed a high activity of CYP3A

in the cells, even higher than in PHH cultured for 48 hours, and importantly, the high activity was stable over

time. CYP3A is involved in the metabolism of many drugs and is therefore of great importance in toxicity and

metabolism studies (Guengerich 1999). The high CYP3A expression in the hiPS-HEP corresponds well to their

sensitivity to the hepatotoxic compounds used in the present study. The hiPS-HEP respond strongly to

amiodarone and alfatoxin B1, both of which are highly dependent on CYP3A activity to generate toxic

metabolites (Elsherbiny et al. 2008; Guengerich et al. 1996). The viability dose response curves display a

significant shift towards lower concentrations during the 7- and 14 days exposure. The other two compounds

tested in this study, troglitazone and ximelagatran behave differently. The hiPS-HEP are sensitive to troglitazone

at higher concentrations and the viability drops from nearly 100% to close to 0% from one concentration to the

next, in the interval of 12.5 µM and 125 µM, making the calculation of the EC10 values difficult. Nevertheless,

the results indicate that the toxicity increases with longer exposure times. We also included ximelagatran, a

direct thrombin inhibitor that was withdrawn from the market because of elevated liver enzymes (ALT-levels)

during long-term clinical trials (Lee et al. 2005). Ximelagatran has been investigated in several pre-clinical

studies without showing an effect on cellular functions, even at concentrations much higher than the plasma

concentration during therapeutic dosing (Kenne et al. 2008). The hiPS-HEP model, despite the possibility of

longer exposure time, does not show a decrease in cell viability at any concentration of ximelagatran tested,

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which is in line with results obtained in other hepatic in vitro systems. The mechanism behind the hepatotoxicity

of ximelagatran is still unresolved and may involve the action of Class II HLA antigens (Kindmark et al. 2008).

We observe a similar sensitivity for HepG2 cells and hiPS-HEP to the compounds during the acute exposure (48

hour time point). A longer incubation time was not possible to perform using the HepG2 cells due to their high

extend of cell proliferation. A comparison to previous studies conducted on PHH indicates a similar ranking of

the sensitivity to the tested compounds. PHH seem most sensitive to aflatoxin B1, followed by amiodarone, and

troglitazone in decreasing order (Chatterjee et al. 2014; Gerets et al. 2012; Szkolnicka et al. 2014). These studies

also demonstrate the large donor-to-donor variability commonly observed with PHH. In addition, the differences

in experimental setups and viability readouts complicate the direct comparison of independent studies.

Nevertheless, Szkolnicka et al. reported an extended compound exposure study in PHH over a 7 day time period,

where an increased sensitivity to amiodarone, similar to our results, was detected. However, in contrast to our

results, they failed to show a toxic effect for troglitazone during the shorter exposure periods (Szkolnicka et al.

2014). The general lack of long-term toxicity studies in PHH illustrates the challenges using this model for long-

term in vitro culturing.

The hiPS-HEP used in this study have several advantages over existing models in that they are generated in a

reproducible way, and are possible to cultivate for long periods of time without loss of hepatic phenotype.

However, they are also limited by their incomplete expression of some drug metabolizing enzymes. Intense

research is ongoing to improve and refine the differentiation process to generate more mature hPSC-derived

hepatocytes. In parallel with these developments and in combination with the possibility of applying prolonged

drug exposure times, hPSC-derived hepatocytes are expected to become an important alternative to PHH in

future drug discovery and toxicity testing. The pharmaceutical industry will most likely benefit from a predictive

cell-based model, with high resemblance to the in vivo situation, which can be used for long-term toxicity

assessment. Such a model can potentially be a complement to existing pre-clinical models, and ultimately allow

earlier testing using human relevant tests, resulting in a reduction of the attritions of candidate drugs entering

phase II and III clinical trials. Another major advantage with the hPSC-based platform for derivation of

functional hepatocytes is the possibility to choose hPSC lines originating from different individuals with diverse

genotypes and having the opportunity to assess compound effects on specifically designed panels of hepatocytes

derived from a variety of donor lines.

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In conclusion, we have performed a thus far, unique long-term chronic toxicity study using hPSC-derived

hepatocytes. The cells show a time-dependent toxic response to amiodarone, aflatoxin B1, and troglitazone. On a

mechanistic level, we also find specific induction of phospholipidosis and steatosis in the cells. Although the

model needs to be challenged with additional toxic compounds, this study demonstrates the utility of hPSC-

derived hepatocytes in long-term toxicity assessment and open up novel avenues for chronic toxicity testing in

vitro.

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Acknowledgements

We thank Jonathan Björklund and Henrik Palmgren, AstraZeneca R&D Mölndal, Sweden, for compound

preparation of Ximelagatran and support with the automated imaging.

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Authorship Contributions

Participates in research design: Holmgren, Sjögren, Andersson, Barragan, Ingelman-Sundberg, Björquist,

Edsbagge

Conducted experiments: Holmgren

Performed data analysis: Holmgren, Sjögren, Sabirsh

Contributed new reagents or analytic tools: Sjögren, Sabirsh, Andersson

Wrote or contributed to the writing of the manuscript: Holmgren, Sartipy, Synnergren, Sjögren, Ingelman-

Sundberg, Björquist, Edsbagge, Andersson, Barragan, Sabirsh

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Footnotes

This study was supported by SCR&Tox, which is funded by the European Commission within its seventh

framework Programme and Cosmetics Europe, the European Cosmetics Association, as part of the SEURAT

(Toward the replacement of in vivo repeated dose system toxicity) Cluster [Contract HEALTH-F5–2010-

266573]; and the Systems Biology Research Centre (University of Skövde, Sweden) under grants from the

Knowledge Foundation [2011/0295].

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Figure Legends

Figure 1. A. Light microscopy image of the hepatocytes morphology (scale bar = 100µm). B. Periodic acid

Schiff staining for glycogen storage (scale bar = 100µm). C. Merged image of Cytokeratin 18 (green) and DAPI

(blue) (scale bar = 50 µm). D. Merged image of HNF4α (red) and DAPI (blue) (scale bar = 50µm).

Figure 2. Viability curves of hiPS-HEP exposed to compounds for 48 hours (red), 7 days (blue), and 14 days

(black) as well as HepG2 exposed to compounds for 48 hours (green). Viability displayed as mean +/- SEM

(n=3).

Figure 3. Hepatotoxicity assessment. A. Cell viability of corresponding cells measured by EZ4U. All samples

were normalized to vehicle control and are presented as mean +/- SEM (n=3). B. Analysis of phospholipidosis,

mean +/- SEM (n=3). C. Analysis of steatosis, mean +/- SEM (n=3). D. Example images from the HCA,

phospholipidosis (red), steatosis (green) and nuclear stain DAPI (blue).

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Tables

Table 1. Study compounds

Compound Top assay concentration

(µM)

Mechanisms of toxicity Cytochrome P450

metabolism

Amiodarone 125 Mitochondrial toxicity

(Kaufmann et al. 2005)

CYP1A1, CYP3A4

(Elsherbiny et al. 2008)

Aflatoxin B1 140 DNA adductor causing

mutations and ROS

formation (Do and Choi

2007)

CYP3A4, CYP1A2

(Guengerich et al. 1996)

Troglitazone 125 Idiosyncratic hepatotoxic,

impaired mitochondria

function (Jaeschke 2007)

CYP3A (Kassahun et al.

2001)

Ximelagatran 400 Unknown hepatotoxic

mechanism, connected to

raised aminotransferase

levels (Lee et al. 2005)

None of the major CYPs

are involved in the

metabolism (Bredberg et

al. 2003; Clement and

Lopian 2003)