studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 535A
655
Regulation of the Hepatic Drug-metabolizing Enzymes
by Probiotics and Conventionalization in Germ-free
Mice
Felcy pavithra Selwyn samraj, Sunny Lihua Cheng, Curtis Klaassen,
Julia Yue Cui; Environmental and Occupational Health Sciences,
University of Washington, Seattle, WA
The use of probiotics and antibiotics has raised concerns of
potential adverse “drug-bacteria” interactions in humans. VSL3
is a probiotic medication that delivers 8 live strains of bacteria
to the host, and has been used to treat ulcerative colitis. The
present study utilized conventional (CV) and germ-free (GF)
mice to determine the effects of VSL3 and conventionalization
of GF mice by exposing them to the CV housing environment
on the expression of approximately 90 critical Phase-I
and –II drug-metabolizing enzymes (DMEs) in liver. Starting at
2-months of age, CV and GF mice (male, n=6-8/group) were
treated with or without VSL3 (4.5×10 6 CFU/ml) in drinking
water for 28 days. In a separate study, 1-month old GF mice
(male, n=4/group) were taken out of the GF isolator and were
housed in the conventional environment for 2 months. Regarding
the bacteria profiles, VSL3 increased each VSL3 component
in the large intestinal content of CV mice, and increased
these bacteria even more in GF mice, likely due to less competition
for growth in the GF-environment (16S rRNA qPCR).
Conventionalization of GF mice increased the total bacteria
and selected bacteria strains with known important functions
for host metabolism in the large intestinal content. Regarding
the effect of VSL3 on the expression of DMEs in livers of CV
mice, VSL3 increased the mRNAs of the Phase-I enzymes cytochrome
P450 (Cyp) 4v3, Alcohol dehydrogenase (Adh) 1,
and Carboxyesterase (Ces) 2a, but decreased the mRNAs
of Cyp3a44 and 3a11; VSL3 also decreased the mRNAs
of multiple Phase-II glutathione-S-transferases (Gstm1-3 and
Gsto1). In livers of GF mice, VSL3 decreased the mRNAs of
the UDP-glucuronosyl transferases (Ugt) 1a9 and 2a3. Regarding
the effects of GF and conventionalization on the hepatic
expression of DMEs, GF mouse livers had increased mRNA of
Ugt1a9, but decreased mRNAs of Gstpi and sulfotransferase
5a1. GF conditions down-regulated many genes in the Cyp3a
cluster but up-regulated many genes in the Cyp4a cluster. Conventionalization
normalized the expression of these genes back
to CV levels. The protein expression of Cyp3a and Cyp4a was
confirmed by Western blot. ChIP-qPCR indicated that changes
in the hepatic PXR- and PPARα-DNA binding to the Cyp3a
and Cyp4a gene clusters during GF- and exGF-conditions
appeared to be responsible for the changes in the Cyp3a and
4a gene expression. In conclusion, alterations in the intestinal
bacteria by VSL3, GF condition, and conventionalization
impact the expression of many DMEs in the host liver, suggesting
the importance of considering “bacteria-drug” interactions
in human populations. (Supported by NIH GM111381,
ES019487, and P30 ES0007033)
Disclosures:
The following authors have nothing to disclose: Felcy pavithra Selwyn samraj,
Sunny Lihua Cheng, Curtis Klaassen, Julia Yue Cui
656
HMGB1 interacts with AIM2 to upregulate hepatocyte
autophagy and prevent cell death after redox stress
Qian Sun, Timothy R. Billiar, Melanie Scott; Surgery, university of
pittsburgh, Pittsburgh, PA
The release of damage-associated molecular patterns such as
double-stranded DNA (dsDNA) after oxidative stress leads
to maturation of the inflammasome and caspase-1 in both
immune and non-immune cells. We have previously shown
that activation of AIM2 inflammasome and caspase-1 in
mouse hepatocytes (HCs) upregulates mitochondrial autophagy
after redox stress rather than leading to the maturation of
cytokines as in immune cells. Nuclear protein HMGB1 binds
dsDNA and translocates to the cytosol during redox stress.
Cytosolic HMGB1 has been shown to upregulate autophagy
to protect against mitochondrial dysfunction. Therefore, we
hypothesized that cytosolic HMGB1 interacts with AIM2, and
its activating ligand dsDNA, to regulate caspase-1 activation
and caspase-1-mediated mitochondrial autophagy in HCs.
Methods: HCs isolated from C57BL/6 (WT) and HC-HMGB1 -/-
(hepatocyte-specific HMGB1 knockout) mice were transfected
with GFP-LC3 before hypoxia (6h, 1% O 2
) and reoxygenation
treatment. Levels of autophagic flux were assessed by increase
in the number of GFP–LC3 puncta in HCs after treatment of
bafilomycin (50nM, 1h). Mitochondrial and cytosolic specific
ROS production was measured by HyPer-Mito and HyPer-
Cyto respectively. Cell death was determined by Annexin V/
PI staining. Interaction between HMGB1 and AIM2 in HCs
was assessed by immunoprecipitation. Results: Hypoxia/reoxygenation
triggered enhanced association between AIM2 and
HMGB1, as well as increased caspase-1 activity in WT HCs,
but not in HC-HMGB1 -/- cells, suggesting a role for HMGB1
in regulating caspase-1 activation. Autophagy levels were
increased in WT HCs after hypoxia/reoxygenation as shown
by increased beclin1 expression and autophagic flux. However,
HC-HMGB1 -/- HCs showed decreased beclin1 levels compared
with WT and impaired autophagic flux after hypoxia/
reoxygenation, similar to our previous results in AIM2 -/- HCs.
Additionally, HC-HMGB1 -/- HCs had increased mitochondrial
volume and ROS production in the mitochondria in comparison
with WT after hypoxia/reoxygenation, suggesting a role for
HMGB1 in up-regulating mitochondrial autophagy. Consistent
with our previous findings in caspase-1 -/- and AIM2 -/- HCs,
HC-HMGB1 -/- HCs showed increased apoptosis and necrosis
in comparison with WT after hypoxia/reoxygenation, confirming
a protective role for HMGB1 in HCs. Conclusion: Our data
suggest that HMGB1 is protective in HCs after redox stress by
upregulating mitochondrial autophagy through its interaction
with AIM2. Our study links HMGB1 with AIM2 inflammasome
activation and stress-induced autophagy in non-immune cells,
which may have implications for future inflammasome-targeting
therapies.
Disclosures:
The following authors have nothing to disclose: Qian Sun, Timothy R. Billiar,
Melanie Scott
657
MYCN is A Novel Biomarker for Chemoprevention of
Hepatocellular Carcinoma by Acyclic Retinoid
Soichi Kojima 1 , Xian-Yang Qin 1 , Harukazu Suzuki 2 , Masao
Honda 3 , Goshi Shiota 4 , Naoto Ishibashi 5 , Hisataka Moriwaki 6 ,
Masahito Shimizu 7 ; 1 Micro-Signaling Regulation Technology
Unit, RIKEN, Wako, Japan; 2 Division of Genomic Technologies,
RIKEN CLST, Yokohama, Japan; 3 Department of Gastroenterology,
Kanazawa University, Kanazawa, Japan; 4 Division of Molecular
and Genetic Medicine, Tottori University, Yonago, Japan; 5 Pharmaceutical
Division, KOWA Company, Higashimurayama, Japan;
6 Gifu University, Gifu, Japan; 7 Department of Gastroenterology,
Gifu University School of Medicine, Gifu, Japan
Background & aim: Poor prognosis of hepatocellular carcinoma
(HCC) is partly due to its high rate of recurrence. Acyclic
retinoid (ACR) is under phase III clinical trials in Japan to