studies

502A AASLD ABSTRACTS HEPATOLOGY, October, 2015
generating higher levels of HCV-expressing AB. Engulfment of
HCV + AB by liver macrophages increases the production of
pro-inflammatory cytokines, thereby exacerbating liver injury
in HCV + alcohol abusers.
Disclosures:
The following authors have nothing to disclose: Murali Ganesan, Larisa Y. Poluektova,
Jinjin Zhang, Tatiana Bronich, Terrence M. Donohue, Kusum K. Kharbanda,
Dean J. Tuma, Natalia A. Osna
590
Mitochondrial Fission Factor (MFF) is induced and binds
to Sab (SH3BP5) in acetaminophen (APAP) and other
JNK dependent hepatotoxicities
Sanda Win, Tin A. Than, Neil Kaplowitz; Medicine, GI/Liver, USC
KSOM, LosAngeles, CA
SAB is a mitochondrial membrane binding target of JNK which
mediates impaired mitochondrial function, increased ROS,
and sustained JNK activation in hepatotoxicity from acetaminophen
(APAP), TNF, palmitic acid, and ER stressors. We have
previously shown that mitochondrial translocation of DRP-1,
the mediator of mitochondrial fission and a key factor in cell
death, is downstream of the interaction of P-JNK and Sab.
MFF is the binding target of DRP-1 in the outer membrane of
mitochondria. Therefore, our aim was to elucidate the role
of Sab in mitochondrial fission. We immunoprecipitated (IP)
Sab from mitochondrial extracts of livers of control and APAP
treated mice (2 hours). MFF was identified in the IP and greatly
increased after APAP, both by western blot and proteomic
analysis. Under basal conditions, low levels of MFF are found
in mitochondria but levels rapidly increased within one hour
after APAP. Proteinase K treatment of intact mitochondria confirmed
that MFF was exclusively in the outer membrane. The
increase in MFF was not inhibited by actinomycin D suggesting
that MFF is stabilized by P-JNK and Sab rather than by induction
of transcription. MFF was also induced in other JNK/Sab
dependent toxicity models such as tunicamycin, palmitic acid
and TNF/galactosamine. Sab knockdown or liver conditional
knockout of Sab abrogated the increase of MFF and prevented
DRP-1 translocation to mitochondria. However, basal levels of
MFF were not altered in the absence of Sab. We previously
showed that knockdown of DOK4, an inner membrane Src
binder, prevented mitochondrial inactivation of Src, mitochondrial
dysfunction, increased ROS, sustained JNK activation,
and cell death from APAP and TNF/galactosamine. Knockdown
of DOK4 had no effect on Sab levels but prevented
the induction of MFF suggesting that P-JNK/Sab mediated
mitochondrial dysfunction leads to increased to MFF levels. In
conclusion, the interaction of P-JNK and Sab leads to induction
of MFF, enhancing mitochondrial fission. MFF appears to be
stabilized by mitochondrial dysfunction and binding to Sab.
Disclosures:
The following authors have nothing to disclose: Sanda Win, Tin A. Than, Neil
Kaplowitz
591
Hormesis in cholestatic liver disease; preconditioning
with low bile acid concentrations protects against bile
acid-toxicity
Esther M. Verhaag, Manon Buist-Homan, Han Moshage, Klaas
Nico Faber; Gastroenterology and Hepatology, University of
Groningen, Groningen, Netherlands
INTRODUCTION: Cholestasis is characterized by accumulation
of bile acids and inflammation, causing hepatocellular damage,
eventually resulting in liver failure. Bile acids induce apoptotic
and necrotic cell death, however, hepatocytes are able to
adapt towards a hostile environment. This suggests a hormetic
response toward bile acids limits hepatocyte cell death during
cholestasis. AIM: To investigate in vitro the mechanisms that
underlie the hormetic response that protect hepatocytes against
experimental cholestatic conditions. METHODS: HepG2.rNTCP
cells were preconditioned (24 h) with sub-apoptotic concentrations
(0.1-50 mM) of various bile acids, the superoxide donor
menadione, the pro-inflammatory cytokine TNF-α or the FXR
agonist GW4064, followed by a challenge with the apoptosis-inducing
bile acid glycochenodeoxycholic acid (GCDCA;
200 mM for 4 h). Levels of caspase-3/7 activity (apoptosis),
cellular LDH leakage (necrosis) and the bile acid exporter BSEP
(ABCB11) mRNA were analyzed. RESULTS: Preconditioning
with the pro-apoptotic bile acids GCDCA or taurocholic acid
(TCA), and the protective bile acids (tauro)ursodeoxycholic
acid (TUDCA, UDCA) reduced GCDCA-induced caspase-3/7
activity in HepG2.rNtcp cells. None of the bile acid-preconditioning
conditions induced cellular LDH leakage from GCD-
CA-challenged HepG2.rNtcp cells above control (untreated
cells) levels. In contrast, preconditioning with sub-apoptotic
concentrations of cholic acid (CA) potentiated GCDCA-induced
apoptosis and similar trends were observed for menadione
and TNF-α preconditioning. CDCA preconditioning did not
change GCDCA-induced levels of caspase-3/7. The hormetic
effect of GCDCA preconditioning was concentration- and
time-dependent, providing significant protection against the
apoptotic GCDCA challenge after at least 12 h preconditioning
with concentrations of GCDCA as low as 1 mM. GCDCA
and CDCA preconditioning both enhanced mRNA levels of
ABCB11. The FXR agonist GW4064 more potently induced
ABCB11 mRNA levels, but did not cause a significant reduction
in GCDCA-induced caspase-3/7 activity, though a trend
towards a partial suppression was observed. CONCLUSIONS:
Sub-toxic concentrations of bile acids in the range that occur
under normal physiological conditions protect HepG2.rNtcp
cells against GCDCA-induced apoptosis, which is largely independent
of FXR signaling. Thus, low concentrations bile acids
enable hepatocytes to withstand sudden increases in bile acid
concentrations and this may explain why only limited levels of
apoptotic cell death is observed in obstructive cholestasis.
Disclosures:
The following authors have nothing to disclose: Esther M. Verhaag, Manon
Buist-Homan, Han Moshage, Klaas Nico Faber
592
A protective role of C/EBP homologous protein in acetaminophen-induced
acute hepatocyte damage in mice
Tsutomu Matsubara 1 , Yuga Teranishi 2 , Kazuki Nakatani 1 , Norifumi
Kawada 2 , Kazuo Ikeda 1 ; 1 Department of Anatomy and
Regenerative Biology, Graduate School of Medicine, Osaka City
University, Osaka, Japan; 2 Department of Hepatology, Graduate
School of Medicine, Osaka City University, Osaka, Japan
Background: Acetaminophen (APAP) overdose causes acute
liver failure as a result of increased oxidative stress followed by
hepatocellular necrosis. C/EBP homologous protein (CHOP)
expression is induced the liver after exposure to APAP, but
the role of CHOP in the APAP-induced liver injury is not fully
understood. This study investigated whether CHOP acts on
the APAP-induced liver injury using wild-type and Chop-null
mice. Methods: Acute liver injury was induced in wild-type
and Chop-null mice by administrating acetaminophen (APAP,
300 mg/kg). The mice were sacrificed at six hours after the
APAP injection. Liver damage was evaluated by measuring
serum ALT levels and liver histology. Hepatic mRNA and pro-