The Toxicologist - Society of Toxicology

1112 HGF ALTERED RESPONSE IN HIGH CHOLESTEROL
FEED MICE HEPATOCYTES.
M. Dominguez, N. Nuño, D. Clavijo, C. Enriquez, V. Souza, L. Bucio, L.
Gomez Quiroz and C. Gutierrez-Ruiz. Health Sciences, Universidad Autónoma
Metropolitana Iztapalapa, Mexico, Mexico.
A high content of cholesterol in the diet can lead to over accumulation deposition
in the liver where it can induce cellular damage,inducing membrane dynamics
changes and improper signaling by receptors.Our objective was to characterize the
oxidative damage induced by an atherogenic diet (2% cholesterol and 0.5 %
sodium cholate) in the hepatocytes and the effect of the HGF as a protective cellular
factor.CD1 mice were fed with an atherogenic diet (HC) for 48 h.Hepatocytes
were isolated following the two-step collagenase perfusion method.Cells were
treated or not with HGF (50 ng/ml.Catalasa, gamma-glutamyl-cysteine synthetase
(γ-GCS),superoxide dismutase 1 (SOD1), phosphor-c-Met and total c-Met content
were assayed by Western blotting;the wound-healing assay was performed at
different times of incubation with HGF. Cholesterol was detected by filliping staining.Protein
oxidation by oxyblot kit.Our data revealed that HC hepatocytes are
under oxidative stress judged by protein oxidation and for the basal content in antioxidant
enzymes such as catalasa,SOD1 and γ-GCS which were increased regarding
chow hepatocytes.Although HGF treatment improve antioxidant enzymes and
protein oxidation,the wound-healing test show that HGF failed in the induction of
repair process in HC hepatocytes.In order to figure out the mechanism for this inappropriate
response elicited by HGF we determined the phosphorylation of the
HGF receptor c-Met.The result show that HGF-induced activation of c-Met in
HC hepatocytes exhibit a delay (max activation 15 min) in comparison with chow
hepatocytes (max activation 1 min,suggesting that cholesterol overload in the hepatocyte
reduces the response of HGF/c-met.Our data show that hepatocytes with an
overload in cholesterol content did not respond adequately to HGF treatment inducing
an insufficient repair perhaps due to c-Met receptor is dependent of a correct
functionality in lipids rafts where cholesterol plays imperative regulatory functions
in signal transduction.
1113 NRF2 ACTIVATION PROMOTES
HYPERCHOLESTEROLEMIA AND GALLSTONE
FORMATION.
M. A. Paranjpe 1 , Q. Cheng 1 , J. Moscovitz 1 , D. Ajay 1 , M. Yamamoto 2 and A.
Slitt 1 . 1 Biomedical and Pharmaceutical Sciences, University of Rhode Island,
Kingston, RI and 2 Medical Biochemistry, Tohoku University, Sendai, Miyagi, Japan.
Cholesterol (CH) gallstone disease is a common gastrointestinal disease and is associated
with consumption of a CH rich diet. Some populations are more susceptible
to gallstone formation, suggesting a genetic component to this susceptibility. The
Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor-E2 related-factor 2
(Nrf2) transcriptional pathway mediates the antioxidant response, but emerging
studies indicate a role for this pathway in modulating hepatic lipid homeostasis.
Preliminary data demonstrate that Nrf2-null mice are resistant to CH gallstone formation
when fed a lithogenic diet (15% fat, 1.25% CH, 0.5% sodium cholate) for
nine weeks. The purpose of this study was to determine whether constitutive Nrf2
activation via Keap1-knock down causes increased CH gallstone formation. Adult
female C57Bl/6 and Keap1-knockdown (Keap1-KD) mice were fed a standard or
lithogenic diet for three weeks and gallbladders, livers and blood were collected.
The lithogenic diet increased CH gallstones in Keap1-KD compared to C57Bl/6
mice. The lithogenic diet increased serum and liver CH levels in both the strains,
but liver CH levels were significantly higher in Keap1-KD mice compared to
C57Bl/6 mice. In addition, the lithogenic diet increased biliary cholesterol concentrations
more in Keap1-KD mice compared to C57Bl/6 mice. Cyp7a1 mRNA expression
in liver was decreased in Keap1-KD mice compared to C57Bl/6 after feeding
a standard or lithogenic diet. Unlike C57Bl/6 mice, Keap1-KD mice failed to
downregulate Hmg-CoA expression in liver after feeding the lithogenic diet. The
lithogenic diet induced the expression of Abcg5, 8 and Abca1 mRNA levels to an
equivalent degree in both the strains of mice. Overall, the data demonstrate that
Nrf2 activation causes increased susceptibility to hypercholesterolemia, which is
most likely due to differential modulation of CH synthesis and metabolism (NIH
5K22ES013782, 3R01ES016042-02S1).
1114 NRF2 PROTECTS MOUSE LIVER FROM OXIDATIVE
INJURY AND STEATOHEPATITIS INDUCED BY
PERSISTENT ACTIVATION OF AHR.
H. Lu and C. D. Klaassen. University of Kansas Medical Center, Kansas City, KS.
Previous studies demonstrate that Nrf2, a master regulator of antioxidative responses,
is essential in mediating the induction of certain antioxidative enzymes by
acute activation of the aryl hydrocarbon receptor (AhR). However, the role of Nrf2
238 SOT 2011 ANNUAL MEETING
in the protection against oxidative stress and DNA damage induced by persistent
activation of the AhR remains unknown, which was investigated in this study. Liver
and blood samples were collected from wild-type (WT) and Nrf2-null mice 21 d
after administration of a bolus low-toxic dose of TCDD 10 μg/kg (ip). After
TCDD treatment, only Nrf2-null mice lost body weight; however, blood levels of
ALT remained essentially unchanged in both genotypes, indicating a lack of extensive
death of hepatocytes. Compared to livers of TCDD-treated WT mice, livers of
TCDD-treated Nrf2-null mice had:1) swollen hepatocytes and markedly higher
hepatic triglycerides; 2) similar induction of Cyp1a1/2, markedly higher expression
of Cyp4a14, but lower catalase; 3) depletion of glutathione, elevation in lipid peroxidation,
DNA damage, and apoptosis; 4) higher induction of proliferative genes
mKi-67 and Top2a; 5) attenuated induction of Nqo1, Gsta2, and Ugt2b35, but
higher induction of Ho-1, Prx-1, p21, and Fgf21; 6) compensatory induction of
Gclc and mEH, 7) higher expression of fatty acid uptake and triglyceride synthetic
genes; 8) higher expression of inflammatory and fibrotic genes; and 9) induction of
c-jun and Gadd45β, a pro-survival gene induced by NF-KB. In conclusion, Nrf2
deficiency markedly increases oxidative stress, DNA damage, and steatohepatitis in
livers with persistent activation of AhR. TCDD activation of AP-1 and NF-KB in
Nrf2-null mice may cause compensatory induction of certain cytoprotective genes
and prevent overt cell death; however, the markedly increased DNA damage and inflammation
in TCDD-treated Nrf2-null mice indicates that Nrf2 deficiency may
substantially increase the risk of liver carcinogenesis during persistent AhR activation
(Supported by NIH grants ES-009716, ES-013714, ES-019487, DK081461,
and RR021940).
1115 TCDD EFFECTS ON HEPATIC LIPID COMPOSITION
IN MICE.
M. M. Angrish 1, 2 , B. D. Mets 2, 3 , D. Wright 3 and T. R. Zacharewski 1, 2, 3 .
1 Genetics Program, Michigan State University, East Lansing, MI, 2 Center for
Integrative Toxicology, Michigan State University, East Lansing, MI and 3 Biochemistry
& Molecular Biology, Michigan State University, East Lansing, MI.
TCDD and related compounds induce steatosis, hepatic vacuolization and hyperlipidemia
in mice. In order to further investigate the effects on hepatic lipid composition,
C57BL/6 female mice were treated with 30 μg/kg TCDD or sesame oil
vehicle for 24, 72 or 168 h. TCDD elicited hepatic vacuolization and inflammation,
and significantly increased relative liver weight (RLW) and hepatic triglyceride
(TRG) levels (1.4-, 2.9- and 4-fold at 24, 72 and 168 h, respectively) compared to
controls. GC-MS analysis of hepatic lipid extracts revealed a TCDD-dependent 2fold
increase in total hepatic fatty acids (TFAs) at 72 and 168 h post dose. More
specifically, TCDD significantly induced long-chain (16C or greater) saturated
fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty
acid (PUFA) levels at 168 h (161, 364 and 218%, respectively). However, there
were significant decreases in the SFA/TFA ratio (25%), with increases in the
MUFA/TFA ratio (167%), and no change in the PUFA/TFA ratio at 168 h. The
reduction in PUFA/MUFA levels (60% at 168 h) was consistent with TCDD-induced
lipid peroxide levels (LPO) (2.5-fold at 168 h). TCDD also induced the essential
fatty acids 18:2n6 and 18:3n3 at all time points, with corresponding decreases
(3-fold at 168 h) in product/precursor ratios (20:4n6/18:2n6 and
20:5n3/18:3n3). These changes can be partially explained by AhR-mediated induction
of Scd1 and Elovl5 (2-4-fold at 24-72 h). Furthermore, computational analysis
from -10,000 bp through the 5’UTR identified putative dioxin response elements
(DRE) and ChIP-chip revealed AhR enriched regions within the Scd1 and
Elovl5 loci. Collectively, TCDD-induced alterations in hepatic SFA, MUFA, PUFA
and LPO levels are consistent with changes in RLW, vacuolization, inflammation
and TRG levels. Gene expression, in silico DRE search and ChIP-chip studies suggest
that these effects are AhR mediated. Funded by SBRP P42ES04911.
1116 PHARMACOLOGIC NRF2 ACTIVATION PROMOTES
HYPERCHOLESTEROLEMIA IN MICE.
J. Moscovitz, M. Paranjpe and A. Slitt. University of Rhode Island, Kingston, RI.
Hypercholesterolemia is associated with consumption of a CH rich diet. Some populations
are more susceptible to hypercholesterolemia, and it is known that there is
a hereditary component to susceptibility. The Kelch-like ECH-associated protein 1
(Keap1)-Nuclear factor-E2 related-factor 2 (Nrf2) transcriptional pathway mediates
the antioxidant response, but emerging studies indicate a role for this pathway
in modulating hepatic lipid homeostasis. The purpose of this study was to determine
whether administration Nrf2 activators affects lithogenic feeding-induced hypercholesterolemia
in mice. Male C57Bl/6 mice were pair-wise fed i) standard diet,
ii) lithogenic diet (LD, 15% fat, 1.25% CH, 0.5% sodium cholate), iii) standard
diet with butylated hydroxyl anisole (BHA, 0.1% w/w) or oleanolic acid (0.1%
w/w), iv) LD with butylated hydroxyl anisole (0.1% w/w) or oleanolic acid (OA,
0.1% w/w) for six weeks. Serum, livers, and gallbladders were collected. BHA and