The Toxicologist - Society of Toxicology

Hepa1c1c7, nor did they enhance the cytotoxicity caused by EL. CoCl 2 was cytotoxic
by itself, but reduced EL cytotoxicity. Results suggest that HIF-1α activation
and signaling is increased in HX/EL-cotreated cells, but the cytotoxic interaction
between HX and EL acts through other signaling pathways. (Supported by R01
ES004139 and T32 ES007255.)
1653 THE ROLE OF MITOCHONDRIA IN AMIODARONE-
INDUCED APOPTOSIS IN RAT PLEURAL
MESOTHELIAL CELLS.
E. S. Taneva and J. M. Cerreta. PHS, St. Johns University, Queens, NY. Sponsor: L.
Trombetta.
Amiodarone is a class III antiarrhythmic agent whose clinical utility may be limited
due to the incidences of amiodarone-induced pulmonary toxicity (AIPT). The
course of AIPT includes alveolitis, phospholipidosis, and irreversible fibrosis. A
number of studies have indicated a key role of epithelial cell apoptosis in the pathogenesis
of AIPT. The purpose of this study was to investigate the cytotoxic effect of
amiodarone (AM) and to elucidate the molecular pathway involved in the AM-induced
apoptotic machinery in rat pleural mesothelial cells (RPMCs). RPMCs were
cultured in Ham’s F-12 medium containing 15 % fetal bovine serum at 37 °C in a
humidified 5 % CO2 environment. The cytotoxicity of AM was tested by using the
methylthiazol diaphenyltetrazolium bromide (MTT) method. Confluent cultures
were exposed to increasing concentrations of the drug (12. 5, 25, 50, and 100
μg/ml) diluted in culture medium over a 24 hour period. All AM concentrations
except for the lowest AM concentration (12.5 μg/ml) significantly decreased the
cell viability. The cytotoxicity was concentration-dependent and the calculated
IC50 value was 50 μg/ml. Additional cultures were pre-treated for 2 hours with the
pan-caspase inhibitor Z-VAD-fmk followed by co-treatment with the IC50 value of
AM and varying concentrations of the inhibitor (1, 5, and 10 μg/ml). Z-VAD-fmk
demonstrated a protective effect on the cell viability at all tested concentrations.
The cells were stained after AM treatment with the cationic fluorophore Rh123 (1
μg/ml for 30 min at 37 °C) to ascertain AM initiates the mitochondria pathway of
the programmed death cascade. AM leads to a decrease in the membrane potentialdriven
accumulation of Rh123 in the inner mitochondrial membrane within 2
hours after treatment with the drug. These data indicate that AM induces cell death
in RPMCs via the mitochondrial apoptotic pathway. Such results may have implications
in understanding the molecular mechanism of AM-induced lung toxicity.
1654 ACUTE SINGLE-MITOCHONDRION RESPONSES TO 1,
3-DINITROBENZENE IN ASTROCYTES.
L. Maurer 1 , I. C. Speirs 1 , J. Fernandez 1 , D. Song 2 , T. Epstein 3 , A. Jureziz 1 , S.
Steiner 1 and M. A. Philbert 1 . 1 Department of Environmental Health Science,
Toxicology, University of Michigan, Ann Arbor, MI, 2 Mechanical Engineering
Department, University of Michigan, Ann Arbor, MI and 3 Chemistry Department,
University of Michigan, Ann Arbor, MI.
As cells in culture age they manifest biochemical changes resulting in loss of function
or cell death. While endpoints such as cell viability are useful, they may not
capture subtle changes that auger changes in cell function. 1,3-Dinitrobenzene
(DNB) is cytotoxic to rat brainstem astrocytes in vivo. Mitochondria in cultured
immortalized astrocytes (DI-TNC1) exposed to DNB exhibit intermittent loss of
inner mitochondrial membrane potential (ΔΨmt) with repolarization prior to permanent
depolarization. Dynamics in DI-TNC1 cells were measured in response to
DNB exposure to investigate early sequelae as a function of cellular ‘age’. To observe
flickering events, DI-TNC1 cells were exposed to DNB (0–600 μM in 0.1%
DMSO) and ΔΨmt was monitored discontinuously using tetramethylrhodamine
methyl ester (100 nM). Cells were maintained in an environmental chamber for up
to 15 minutes and imaged using an Olympus IX81. Original micrographs were
converted to binary images in MatLab. Mean mitochondrial aspect ratios were calculated
and modeled as a function of DNB concentration, exposure time, and cell
passage (cellular age). Cells exposed to DNB exhibited a negative, age-dependent
relationship between concentration and time to initial flickering events. These
events were statistically significant (p