The Toxicologist - Society of Toxicology

at a range of concentrations in a variety of tissue types. Despite this, alternative programmed
cell pathways induced by arsenic have been proposed. Autophagy is involved
in the degradation of damaged organelles and cellular proteins, and can also
play a role in determining the fate of stressed cells. Human lymphoblastoid cell
lines (LCL) have been used as in-vitro models in arsenic toxicology for many years,
but the specific mechanism of arsenic-induced cytotoxicity in LCL is still unknown.
We investigated the mechanism of sodium arsenite cytotoxicity in LCL
18564 using a combination of markers for cell death pathways. Apoptotic markers
(phosphatidylserine externalization, PARP cleavage, and response to caspase inhibition)
were all negative in arsenite-exposed cells. In contrast, electron microscopy
images and acidophilic dye fluorescence data indicated that autophagy is induced
by arsenite and is correlated with arsenite-induced cytotoxicity in this LCL. It is unclear
whether autophagy is a compensatory mechanism induced by arsenite exposure
in LCL, or alternatively a cell death-effecting process in LCL. The ability of arsenite
to modulate the autophagy pathway in LCL lends insight into the molecular
mechanism of arsenite-induced cytotoxicity and suggests novel biologic targets in
arsenic toxicology. (Funded by ES 006694, ES 16652, and ES 04940)
1228 RAPTOR, A COMPONENT OF MTORC1, PLAYS A KEY
ROLE IN AUTOPHAGY IN ADAPTION OF
RADIORESISTANCE IN A549 LUNG CANCER CELLS.
J. Shin and M. Cho. Lab. of Toxicology, College of Veterinary Medicine, Seoul
National University, Seoul, Republic of Korea.
Autophagy has been a sensational topic among oncologists recently, in that it plays
a dual role – survival or death, depending on cellular circumstances. While incidence
as well as mortality figures for lung cancer increase, the importance of controlling
therapy-induced resistance rises accordingly. As radiotherapy-induced resistance
in cancer cells has found to be mTOR-dependent, many approaches are
made to enhance therapeutic efficacy by applying mTOR inhibitors to make the
target cells to be more radiosensitive. mTORC1, consists of Raptor, mLST,
PRAS40, is an upstream negative regulator of autophagy, which works in a complex,
however, the role of each components has not yet been clarified.
To test the basic role of raptor in gamma-irradiated A549 cancer cells, raptor-stably-overexpressed
(A549-Raptor) and raptor-stably-downregulated (A549-shRNA
Raptor) cell line were established and compared. As lysosome is a final destination
for autophagic/lysosomal protein degradation, effects of raptor expression towards
autophagy was examined. Expression of proteins involved in the formation and
maturation of autophagosomes (beclin-1, LC3, Atg5) or associated with autolysosomes
and lysosomes (LAMP-1, cathepsin D), as well as downstream proteins of
mTOR (p70S6K, eIF4E) were determined. Interestingly after 72 hours of gammairradiation
p70S6K was totally degraded in A549, but no change was shown in
A549-Raptor, indicating that raptor might have an effect in radioresistance. Further
observation was made with treatment of chloroquine, the lysosomal membrane
destabilizer – autophagy inhibitor, and increase of beclin-1 and procathepsin D,
but not mature cathepsin D, and a decrease in expression of LAMP-1 were noticed.
Taken together, control of raptor expression in cancer cells can be suggested to be a
novel therapeutic approach to overcome radioresistance after radiotherapy and to
increase radiosensitivity when combined with chemotherapy.
1229 MECHANISMS OF AMIODARONE AND
DESETHYLAMIODARONE CYTOTOXICITY IN
HUMAN LUNG CELLS.
J. E. Black, J. F. Brien, W. J. Racz and T. E. Massey. Queen’s University, Kingston,
ON, Canada.
Amiodarone (AM) is a potent antidysrhythmic agent which can cause potentially
life-threatening pulmonary fibrosis, and desethylamiodarone (DEA) is a metabolite
of AM that may contribute to the toxicity of AM in vivo. Recent evidence has implicated
the involvement of the renin-angiotensin system (RAS) in the initiation
and progression of amiodarone-induced pulmonary toxicity (AIPT). In HPL1A
human peripheral lung epithelial cells, we found AM to be converted to DEA minimally
(