Investigations into idiosyncratic drug-induced
hepatotoxicity and chronic proliferation of cancer cells
using a label-free method
Corina Bot3, Sonja Stölzle-Feix1, Elena Dragicevic1, Krisztina Juhasz1,2, Leo Doerr1, Matthias Beckler1, MichaelGeorge1, Andrea Brüggemann1, Rodolfo Haedo3, Niels Fertig1
1Nanion Technologies, Munich, Germany, 2Technical University of Munich, Germany, 3NanioTechnologies Inc.,

Livingston, NJ, 07039 (USA)Introduction
Hepatic toxicity has accounted for 15 of the 47 drugs withdrawn from themarket in the last two decades. More specifically, Drug Induced Liver Injury(DILI) is the major cause of acute liver failure in the USA and Europe and is
one of the main reasons for regulatory actions. DILI is classified as intrinsic(or dose-dependent) or as idiosyncratic. A prominent example ofidiosyncrasy is acetaminophen (paracetamol), with a variable time ofonset and not directly dependent on dose. We present a non-invasive DILI
assay approach based on impedance measurements in monocytederived hepatocyte-like (MH) cells from MetaHeps®.

Cell proliferation monitored via impedanceMH cells were used on a 96-well screening system that monitors changes inimpedance (CardioExcyte 96). Once the monolayer is exposed to acytotoxic agent, the impedance changes and measures of toxicity can bequantified long-term. We investigated the hepatotoxic effects ofparacetamol on MH cells when exposed for 24 and 48 hours. In agreementwith other standard toxicity assays, such as the lactate dehydrogenase
release assay, low doses of paracetamol caused transient toxicity and‘adaptation’ was observed. At higher doses, hepatotoxic effects ofparacetamol could be reversed upon washout after 24 hours, butcontinued exposure caused increased hepatotoxicity.

Left: Averaged relative changes of the impedance signal withcorresponding standard deviation for CHO cells with differing initial celldensity (as indicated) over a cell proliferation time period of 72 h (n = 24;left). Data are normalized to medium reference without cells and cell
addition time point.

In addition to hepatotoxicity, another validation of the principle is shown forchronic proliferation of cancer cells. Traditional cell proliferation assays
involve labeling cells of interest with compounds that become reduced in
the environment of metabolically active cells, or by incubating cells withradioactive labels.

Right: Effect of Escin on the impedance signal of CHO cells seeded withinitial cell density of 16 k cells/well in concentrations of 6.8 μM, 13.6 μM, 27.2
μM and 54.4 μM (n = 4; right). Effect of Triton X-100 (2 %) is also shown. Dataare normalized to the respective compound reference without cells andthe compound addition time point.

Investigating therapy resistance of cancer cells in vitroHepatotoxicity of paracetamol

Left: Increasing concentrations of paracetamol induce a decrease inbase impedance of monocyte-derived hepatocyte-like (MH) cells which
can be monitored continuously (left). Tween (2%) induced 100% cell
death and was used as a positive control.
Right: Normalized impedance versus time (in hours) after MH cells were
exposed to 5 mM paracetamol for 24 or 48 hours. Cells recovered,indicated by the increase in base impedance, when paracetamol waswashed out after 24 hours but toxicity continued when 5 mM
paracetamol was added again for a further 24 hours. This is consistent
with data obtained using the LDH release assay which shows that toxicity
is reversed upon washout of paracetamol but continues with a 2nd doseof paracetamol after 24 hours (data not shown).

Conclusions

In this study, murine mammary carcinoma cells (H8N8 and H8N8 T3.2) were used on the CardioExcyte 96 and monitored over a time period of 500 h. Murine mammary
carcinoma cells (H8N8 and H8N8 T3.2) were used and changes in impedance, and therefore confluency, were used as a measure of toxicity. Intrinsic (dosedependent) effects of the standard clinical treatment regimen cyclophosphamide, doxorubicin and 5-fluouracil could be identified consistent with other methods oflive cell analysis systems.

Impedance

2.0• Cytotoxic effects of Escin and Triton X can be detected using impedance
measurements of proliferating cells• The hepatotoxic effects of paracetamol on hepatocyte-like cells(MetaHeps) are reversible upon removal of paracetamol after 24 hours
but remain upon chronic exposure

1.5• Data comparable to LDH studies but with continuous monitoring possible
40 x

1.0200Time/h400

Left: Increasing CAF* concentrations showed a dose dependent effect on the viability of H8N8 cancer cells. Re-growth of cells after washout of the standard clinicaltreatment regimen was dependent on the previously applied dose. Middle: H8N8 cancer cells at 40 x magnification. Right: Transparent CardioExcyte 96 sensor plates
allow imaging and optical visualization of seeded cells.

• The 96-well impedance system CardioExcyte 96 in combination withmurine mammary carcinoma cells provides a novel tool for investigating
therapy resistance of cancer cells in vitro.
• The label-free CardioExcyte 96 impedance platform enables acute andchronic assessment of toxicity in a continuous fashion from living cells
without the confounding effects of dyes that may affect cell function