The Toxicologist - Society of Toxicology

associated with anti-cancer agents, in vitro approaches are key investigative tools.
iCell® cardiomyocytes (Cellular Dynamics International, Madison, WI) are highly
purified human cardiomyocytes derived from induced pluripotent stem cells
through reprogramming adult cells, and are becoming widely used for the assessment
of cardiac toxicity. To begin to inform the utility of these cells to monitor cellular
events involved in critical cardiac myocyte signaling, we measured expression
and activity of ErbB receptors, Erk1, Erk2, AKT, troponins, and endothelin-1 (ET-
1) receptors A and B. Untreated iCell® cardiomyocytes expressed ErbB2 and
ErbB4 receptors, Erk1, Erk2, AKT, cardiac troponin I, cardiac troponin T and ET-
1 receptors A and B, but not ErbB1 or ErbB3 receptors. Furthermore, treatment
with neuregulin-1 (a natural ligand for ErbB4) activated ERK1/2 and AKT as
demonstrated by increased levels of phosphorylated proteins measured by western
blotting with phospho-specific antibodies. Ligand-activated ErbB2/ErbB4 signaling
was both dose and time dependent. We also examined the effect of tyrosine kinase
inhibitors (lapatinib, sorafanib and staurosporine) and anthracyclines (doxorubicin)
on cell death and mitochondrial membrane potential (MMP) using a high
content multiplexed imaging system, and on troponin release into the cell culture
media using the Meso Scale Discovery® platform. All of these anticancer agents induced
a dose-dependent release of cardiac troponins I and T and caused a reduction
in MMP. Based on these results, this model may present an opportunity to explore
and “back-translate” mechanisms of cardiotoxicity using human cardiomyocytes in
vitro. Funded by NCI Contract No HHSN261200800001E.
80 Mitochondria and Intrinsic Apoptotic Pathway Mediate
Cardiac Toxicity of Environmentally Persistent Free Radicals
(EPFR).
G. C. Chuang 1 , B. Dellinger 2 and K. J. Varner 1 . 1 Pharmacology, Louisiana State
University Health Sciences Center, New Orleans, LA; 2 Chemistry, Louisiana State
University and A&M College, Baton Rouge, LA.
Epidemiology studies have linked combustion-derived particulate matter to increased
cardiac morbidity and mortality. To conduct prospective controlled-exposure
studies, we synthesized the model EPFR, DCB230, by chemisorption of 1,2dichlorobenzene
to 0.2 μm silica particles containing 5% Cu(II)O at 230°C.
Electron paramagnetic resonance showed that DCB230 generates radicals in solution
similar to those produced by EPFRs found in environmental samples. We have
shown that DCB230 inhalation produces inflammation and oxidative stress in
both lung and heart. Since oxidative stress and apoptosis are mechanisms implicated
in cardiac injury, we hypothesized that DCB230 exposure exerts cardiotoxicity
by activating apoptotic pathways. HL-1 cardiomyocytes were dosed with 0-200
μg/mL DCB230 for 8 h and assessed for cytotoxicity and apoptotic markers.
DCB230 dose-dependently increased lactate dehydrogenase (LDH) release, a
marker of late cell death. Caspase 3, caspase 9, and poly (ADP-ribose) polymerase 1
cleavage also increased concomitant with DCB230 concentration, indicating apoptotic
signaling activation. Next, HL-1 cardiomyocytes were treated with 0-200
μg/mL DCB230 for 2 h to examine early signaling events. While neither LDH release
nor caspase 3 cleavage were detected after 2 h, increased caspase 9 cleavage
suggests that mitochondrial dysfunction initiated early intrinsic apoptotic signaling.
Confocal microscopy showed that mitochondrial membrane potential decreased
after 2 h treatment with DCB230. Lastly, fluorescence data was validated as
mitochondrial via FRET and co-localization with MitoTracker Green. Taken together,
DCB230 exposure depolarized mitochondria in cardiomyocytes, leading to
the activation of canonical intrinsic apoptotic signaling and resulting in cell death.
Future studies will assess mitochondrial permeability transition and autophagy as
contributing mechanisms. Supported by NIH P42-ES013648, sub-award 61365.
81 Evaluation of Cellular Impedance Assays for Drug Screening
in Cardiomyocytes.
M. Peters, C. W. Scott, S. D. Lamore and Y. P. Dragan. Safety Assessment,
AstraZeneca, Waltham, MA.
Cardiovascular (CV) toxicity is a leading contributor to drug withdrawal and latestage
attrition. Earlier screening is a validated approach to build-in CV safety, as
demonstrated for hERG screening to reduce arrhythmia. There is an urgent need
for novel in vitro assays to extend this success to contractility, heart rate, hypertrophy,
structural damage, and non-hERG arrhythmia. Advances in cellular impedance
technology enables label-free tracking of spontaneous synchronized beating of
cultured cardiomyocytes (CM). To validate and translate CM impedance assays, we
tested a set of drugs with established CV effects in humans- 22 neg. inotropes, 8
pos. inotropes, and 21 inactives (previously tested in canine CM Tox Appl Pharm
260(2):162). The data clearly indicate that beat rate and amplitude are independent
variables, capable of providing robust potency data. Consistent with the balance
of negative inotropes, the most frequent response was a dose-dependent decrease in
16 SOT 2013 ANNUAL MEETING
amplitude until beating stopped. The cessation of beating was not linked to cytotoxicity
(judged by ATP and cell index) indicating specific changes in CM function.
Since rat neonatal (and stem cell-derived) CMs have a negative frequencyforce
relationship, it is not surprising that the decrease in amplitude was linked to a
concomitant increase in rate. However, for another subset of validation compounds,
rate initially decreased, whereas amplitude showed no associated change
until higher drug concentrations. Moreover, for a test compound not in the validation
set (that was selected for inducing myocarditis in 2 days), beat rate increased
with no change in amplitude or cytotoxicity. Together this data demonstrates that
impedance assays can detect and differentiated functional changes in CMs. The
changes are sensitive to electrical and mechanical aspects of contraction, yield robust
data, and offer a versatile format with moderate throughput making this platform
a candidate for addressing gaps in early phase screening for CV toxicity.
82 Cellular Impedance Assays for Predictive Preclinical Drug
Screening of Kinase Inhibitors in Cardiovascular Toxicity.
S. D. Lamore, C. W. Scott, Y. P. Dragan and M. Peters. Safety Assessment,
AstraZeneca, Waltham, MA.
Cardiotoxicity is the leading cause for late stage drug attrition and withdrawals
from the market. Serious adverse cardiac events have emerged as a prevalent risk for
kinase inhibitors (KI). Although current in vivo screens can reduce known risks due
to arrhythmia, there is urgent need for novel in vitro assays with sufficient throughput
to identify risks and support the development of SAR against other prevalent
cardiovascular (CV) toxicities. Recently, cellular impedance technology has been
adapted for detecting spontaneous, synchronized beating of cultures of cardiomyocytes
(CM) in a real-time, label-free format. Impedance technology is a good candidate
for detecting the pleiotropic cellular effects of kinases since it detects morphological
changes thereby giving a readout that is downstream of key toxicity
targets in the contraction cascade (i.e. cardiac action potential, calcium flux, mechanical
elements of contraction). We evaluated the application of impedancebased
assays for screening KI effects on rat neonatal CM. We selected compounds
from a MAP-microtubule affinity-regulating kinase (MARK) inhibitor program
that failed in late-stage preclinical development with dramatically decreased blood
pressure in anesthetized dogs as an example for the earlier detection of CV toxicity.
Two MARK inhibitors were tested and both dose-dependently influenced CM beat
rate and amplitude without causing cell death as judged by cell index, cellular ATP
levels, or cardiac troponin release assays. The relative potency of the two compounds
on reducing beat amplitude in impedance assays (EC50= 4.31μM and 0.55
μM; 20 min exposure) aligned with the ~10-fold difference in affinity for MARK
isoforms 1-4. Knockdown of pan-MARK expression reduced beat amplitude by
40%. These MARK data indicate that impedance assays can specifically detect noncytotoxic
functional effects of KIs on CM. Our data support the validation of cellular
impedance-based assays for an early preclinical CV toxicity screening of KIs.
83 Activation of Human Monocytic NADPH Oxidase by
Chlorinated Cyclodiene Insecticides.
L. C. Mangum, J. E. Chambers and M. K. Ross. CVM Basic Sciences, Mississippi
State University, Mississippi State, MS.
Although the mechanistic relationship between bioaccumulative organochlorine
(OC) insecticide exposure and increased atherosclerosis risk is poorly defined, elevated
systemic oxidative stress stemming from OC-mediated induction of NADPH
oxidase activity may play a significant role in disease development. Activation of
phagocytic Nox2-containing NADPH oxidase can result in a rapid intracellular accumulation
of superoxide-derived reactive oxygen species (ROS) that may be directly
linked to the progression of atherosclerosis. This study measured the ability of
two legacy OC compounds to induce Nox2-containing NADPH oxidase activity in
vitro, in addition to providing evidence for a possible mechanism of action. Human
THP-1 monocytes exposed to micromolar amounts (1-20 μM) of the cyclodiene
OC insecticides trans-nonachlor and dieldrin exhibited increased levels of serine
phosphorylation of the p47phox regulatory subunit of NADPH oxidase, a necessary
process for enzyme assembly and translocation, with trans-nonachlor demonstrating
several fold greater potency than dieldrin. OC treatment also induced elevated
levels of intracellular ROS, as shown by 2’,7’- dichlorofluorescein-diacetate
fluorescence assay, suggesting increased superoxide anion production. Pretreatment
of monocytes with arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic
phospholipase A2, prior to cyclodiene treatment abrogated p47phox serine
phosphorylation and blocked the induction of arachidonic acid and prostanoid liberation,
as determined by UPLC-ESI MS/MS, suggesting that this enzyme may
play a crucial role in the induction of NADPH oxidase activity by cyclodienes via
the modulation of intracellular arachidonic acid levels. The results suggest that
trans-nonachlor and dieldrin are capable of altering intracellular ROS levels via an