The Toxicologist - Society of Toxicology

1662 INDUCTION OF APOPTOSIS-ASSOCIATED
RIBOSOMAL RNA (RRNA) CLEAVAGE BY THE
TRICHOTHECENE DEOXYNIVALENOL.
K. He 1, 2 , H. Zhou 3 and J. Pestka 1, 2, 3 . 1 Department of Microbiology & Molecular
Genetics, Michigan State University, East Lansing, MI, 2 Center for Integrative
Toxicology, Michigan State University, East Lansing, MI and 3 Food Science &
Human Nutrition, Michigan State University, East Lansing, MI.
The trichothecene deoxynivalenol (DON) can bind to the ribosome, inhibit protein
translation and induce rRNA cleavage. However, the signaling pathway leading
to rRNA cleavage is still unknown. Our laboratory previously found that DON activates
of mitogen-activated protein kinases (MAPKs) which mediate both gene expression
and apoptosis and that both double-stranded RNA activated kinase (PKR)
and hematopoietic cell kinase (Hck) are critical upstream kinases for these effects.
Selective inhibitors were used to elucidate the mechanism of DON-induced rRNA
cleavage in the RAW 264.7 macrophage model. RAW cells (5×106/10cm dish)
were cultured overnight and pre-treated with inhibitors 1 h followed by incubation
with DON (1000 ng/ml) for 6 h. Both the PKR inhibitor C-16 (>0.1μM) and Hck
inhibitor PP1 (>25μM) inhibited rRNA cleavage. When inhibitors for p38, JNK
and ERK were tested, only the p38 inhibitor (>5 μM) impaired rRNA cleavage. In
addition, the general caspase inhibitor Z-VAD-FMK (>100uM) suppressed rRNA
cleavage suggesting that this effect is apoptosis-associated. Pifithrin-α which can reversibly
inhibit p53-dependent transactivation of p53-responsive genes and apoptosis,
inhibited rRNA cleavage at 100 μM. Similarly, pifithrin-μ, which blocks p53
interaction with Bcl-2 family proteins and selectively inhibits p53 translocation to
mitochondria completely, suppressed rRNA cleavage at 25μM. Thus DON-induced
rRNA cleavage appears to be closely linked to the intrinsic apoptotic pathway
sequentially involving PKR-Hck-p38-p53-caspase activation.
1663 DEVELOPMENT OF NOVEL INDOLEQUINONE ANTI-
TUMOR AGENTS: MECHANISM OF TOXICITY IN
HUMAN PANCREATIC CANCER.
C. Yan 1 , D. Siegel 1 , M. Colucci 2 , A. Chilloux 2 , J. Newsome 2 , C. J. Moody 2
and D. Ross 1 . 1 Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO
and 2 Chemistry, University of Nottingham, Nottingham, United Kingdom.
A series of novel indolequinones was developed as potent antitumor agents against
human pancreatic cancer. Three general classes of indolequinones with varying substitutions
at the indole 2- position exhibited marked growth inhibitory activity
against human pancreatic cancer both in-vitro (MTT and clonogenic assays) and
in-vivo (mouse xenograft models). The pancreatic cancer cell lines PANC-1, MIA
PaCa-2, and BxPC-3 were used as in-vitro model systems and the IC50 values of
the indolequinones in all three cell lines were in the low nanomolar range.
Indolequinones were also found to be efficient inducers of apoptosis in these cell
lines at concentrations which induced growth inhibition. Selected indolequinones
were screened against the NCI-60 cell line panel and their spectrum of activity was
similar to established inhibitors of thioredoxin reductase. Indolequinones were
therefore tested as inhibitors of thioredoxin reductase and were found to inhibit the
enzyme in pancreatic cancer cells at concentrations equivalent to those inducing
growth inhibitory effects. The mechanism of inhibition of thioredoxin reductase by
the indolequinones was then studied in detail in cell-free systems using purified enzyme
and the C-terminal selenocysteine of thioredoxin reductase was found to be
the primary adduction site of the indolequinones using LC-MS analysis. Inhibition
of thioredoxin reductase by indolequinones in pancreatic cancer cells resulted in a
change of thioredoxin redox state and activation of the p38/JNK signaling pathway.
Oxidized thioredoxin is known to activate apoptosis signal-regulating kinase 1
(ASK1), the upstream activator of p38/JNK in the MAPK signaling cascade, providing
a potential mechanism for indolequinone-induced apoptosis. Our results
demonstrate that thioredoxin reductase, p38 and JNK, are potential targets of antitumor
indolequinones in pancreatic cancer cells (Supported by CA11441).
1664 SDF-1β PROTECTS CARDIAC CELLS FROM
PALMITATE-INDUCED NITROSATIVE STRESS-
MEDIATED ER STRESS AND CELL DEATH THROUGH
ACTIVATION OF AMPK-MEDIATED IL-6 EXCRETION.
Y. Zhao 2, 1 , W. Li 2 and L. Cai 1, 2 . 1 University of Louisville, Louisville, KY and 2 The
Fist Hospital of Jilin University, Changchun, China.
Elevated saturated free fatty acids are trigger for cardiac cell death,but its mechanisms
remain unknown. Stromal cell derived factor-1beta(SDF-1β)was found to
play cardiac protection,whether it protects the cardiac cells from lipotoxicity remains
unknown.Using H9c2 cell line,the present study was undertaken to investi-
358 SOT 2011 ANNUAL MEETING
gate apoptotic effect of Pal and possible protective effect of SDF-1β. Exposureof
H9c2 cells to Pal at 62.5μM for 15h causes a significant apoptotic effect. This effect
is mediated by endoplasmic reticulum (ER) stress associated cell death that is
caused by NADPH oxidase (NOX)-activation associated nitrosative stress since Pal
induces significant increases in 3-nitrotyrosine at 3–9h and ER stress at 6–9h after
treatment.Inhibiting NOX-activation or scavenging superoxide and peroxynitrite
with their specific inhibitors or scavenger abolishes Pal-induced ER stress and cell
death.Inhibiting ER stress prevents cell death. Pretreatment with SDF-1β abolishes
Pal-induced nitrosative damage,ER stress and cell death,along with up-regulation
of AMPK-mediated IL-6 production. Direct addition of AMPK activator significantly
prevents Pal-induced cell death and also increases IL-6 contents while inhibition
of AMPK abolishes SDF-1β-mediated cardiac protection. To further define
the role of AMPK-mediated IL-6 in cardiac protection,direct addition of recombinant
IL-6 to cell cultures offers a significant prevent of Pal-induced ER-stress and
cell death.These results suggest that cardiac apoptotic effect of Pal is mediated by
NOX activation-triggered nitrosative damage and ER stress cell death
pathway.Pretreatment with SDF-1β can significantly prevent Pal-induced cardiac
cell death via activation of AMPK-mediated IL-6 to directly protect from cell
death via inhibition of ER-stress.The finding that SDF-1β protects cardiac cell
death from fatty acid via up-regulation of AMPK opens a new road for the research
on cardiac protection by SDF-1β that provides cardiac protection independent of
angiogenesis.
1665 THE MECHANISMS OF PARAQUAT-INDUCED LUNG
TOXICITY IN VIVO AND IN VITRO.
Y. Yang 1 , D. Hung 1, 2 , C. Chen 1, 3 , H. Wu 4 , K. Chen 4 , C. Su 5 , C. Huang 6 and
Y. Chen 7 . 1 Graduate Institute of Drug Safety, China Medical University, Taichung,
Taiwan, 2 Toxicology Center, China Medical University Hospital, Taichung, Taiwan,
3 Department of Emergency, China Medical University Hospital, Taichung, Taiwan,
4 Department of Urology, China Medical University Hospital, Taichung, Taiwan,
5 Department of Otorhinolaryngology, Head and Neck Surgery, Changhua Christian
Hospital, Changhua, Taiwan, 6 School of Chinese Medicine, China Medical
University, Taichung, Taiwan and 7 Department of Physiology, China Medical
University, Taichung, Taiwan.
Paraquat (1,1’-dimethyl-4,4’-bipyridium dichloride) is an effective herbicide which
is used all over the world. Paraquat induces fatal damage to multiple organs, especially
lung. The mechanisms of cytotoxicity of paraquat are redox cycling and intracellular
oxidative stress generation. In this study, we investigated the effects and
the possible mechanisms of paraquat on the rat lung alveolar epithelial cell line (L2)
and mouse lung tissue. In our study, L2 cells were exposed to paraquat
(50–900μM) for 24 h. These results showed that cell viability were decreased, malondialdehyde
(MDA) and ROS levels were increased in a dose-dependent manner
after paraquat exposure for 24 h. Besides, paraquat increased proteins of Bax, cytosol
apoptosis-inducing factor (AIF), cytosol Endonuclease G (Endo G) expression,
and enhanced mitochondrial membrane potential depolarization.
Furthermore, mitochondria-related apoptotic signals of mRNAs of Bax, Bid, p53,
mdm-2 were increased in L2 cell after exposure to paraquat. In addition, mRNAs
levels of endoplasmic reticulum stress (ER stress) related apoptotic signals of
ATF4,ATF6,GRP78,GRP94,CHOP were significantly increased. In vivo, the
MDA levels in plasma and lung tissues were dramatically raised after paraquat
treatment, but these effects were reversed by antioxidant, N-acetyl-L-cysteine
(NAC). These results suggest that paraquat induced alveolar epithelial cell death
through a mitochondria- and ER stress-related pathway. These observations provide
evidence that paraquat is an environmental risk factor for lung functional disorders
and diseases.
1666 ROLE OF CANNABINOID RECEPTORS IN
CANNABIDIOL-MEDIATED APOPTOSIS IN
LIPOPOLYSACCHARIDE(LPS) ACTIVATED B
LYMPHOCYTES.
R. Rao, P. Nagarkatti and M. Nagarkatti. University of South Carolina,
Columbia, SC.
Cannabinoids are biologically active components of marijuana (Cannabis sativa).
Cannabinoids mediate their effects via the Cannabinoid receptors. Cannabinoid receptor
1 (CB1), although predominantly expressed in the brain, is also expressed in
the cells of immune origin. Cannabinoid receptor 2 (CB2) however is primarily expressed
in immune cells, with B lymphocytes expressing it in highest amounts.
Cannabidiol (CBD) is a major non-psychoactive component of marijuana that exhibits
anti-inflammatory properties. In the current study we investigated the effects
of CBD on LPS activated B lymphocytes specifically its ability to induce apoptosis.
We found that CBD at a concentration of 10-20 μM caused significant levels of
apoptosis in CD19 positive B cells in vitro. B cells from CB1 knock out (KO) mice