Plasma microRNAs are sensitive indicators of inter-strain differences in the severity of liver injury induced in mice by a choline- and folate-deficient diet

Article (PDF Available)inToxicology and Applied Pharmacology 262(1):52-9 · April 2012with36 Reads
DOI: 10.1016/j.taap.2012.04.018 · Source: PubMed
Abstract
MicroRNAs (miRNAs) are a class of small, conserved, tissue-specific regulatory non-coding RNAs that modulate a variety of biological processes and play a fundamental role in the pathogenesis of major human diseases, including nonalcoholic fatty liver disease (NAFLD). However, the association between inter-individual differences in susceptibility to NAFLD and altered miRNA expression is largely unknown. In view of this, the goals of the present study were (i) to determine whether or not individual differences in the extent of NAFLD-induced liver injury are associated with altered miRNA expression, and (ii) assess if circulating blood miRNAs may be used as potential biomarkers for the noninvasive evaluation of the severity of NAFLD. A panel of seven genetically diverse strains of inbred male mice (A/J, C57BL/6J, C3H/HeJ, 129S/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were fed a choline- and folate-deficient (CFD) diet for 12weeks. This diet induced liver injury in all mouse strains; however, the extent of NAFLD-associated pathomorphological changes in the livers was strain-specific, with A/J, C57BL/6J, and C3H/HeJ mice being the least sensitive and WSB/EiJ mice being the most sensitive. The morphological changes in the livers were accompanied by differences in the levels of hepatic and plasma miRNAs. The levels of circulating miR-34a, miR-122, miR-181a, miR-192, and miR-200b miRNAs were significantly correlated with a severity of NAFLD-specific liver pathomorphological features, with the strongest correlation occurring with miR-34a. These observations suggest that the plasma levels of miRNAs may be used as biomarkers for noninvasive monitoring the extent of NAFLD-associated liver injury and susceptibility to NAFLD.
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Plasma microRNAs are sensitive indicators of inter-strain differences in the severity
of liver injury induced in mice by a choline- and folate-decient diet
Volodymyr P. Tryndyak
a
, John R. Latendresse
b
, Beverly Montgomery
a
, Sharon A. Ross
c
,
Frederick A. Beland
a
, Ivan Rusyn
d
, Igor P. Pogribny
a,
a
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA
b
Toxicologic Pathology Associates, National Center for Toxicological Research, Jefferson, AR 72079, USA
c
Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA
d
Department of Environmental Sciences & Engineering, University of North Carolina, Chapel Hill, NC 27599, USA
abstractarticle info
Article history:
Received 5 March 2012
Revised 5 April 2012
Accepted 16 April 2012
Available online 24 April 2012
Keywords:
Inter-individual differences
microRNAs
Mouse
Nonalcoholic fatty liver disease
MicroRNAs (miRNAs) are a class of small, conserved, tissue-specic regulatory non-coding RNAs that modulate
a variety of biological processes and play a fundamental role in the pathogenesis of major human diseases, in-
cluding nonalcoholic fatty liver disease (NAFLD). However, the association between inter-individual differences
in susceptibility to NAFLD and altered miRNA expression is largely unknown. In view of this, the goals of the pre-
sent study were (i) to determine whether or not individual differences in the extent of NAFLD-induced liver
injury are associated with altered miRNA expression, and (ii) assess if circulating blood miRNAs may be used
as potential biomarkers for the noninvasive evaluation of the severity of NAFLD. A panel of seven genetically di-
verse strains of inbred malemice (A/J, C57BL/6J, C3H/HeJ, 129S/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ)were fed
a choline- and folate-decient (CFD) diet for 12weeks. This diet induced liver injury in all mouse strains; how-
ever, the extent of NAFLD-associated pathomorphological changes in the livers was strain-specic, with A/J,
C57BL/6J,and C3H/HeJ mice being the leastsensitive and WSB/EiJ micebeing the most sensitive. The morpholog-
ical changesin the livers were accompaniedby differences in the levels of hepatic and plasma miRNAs. The levels
of circulating miR-34a, miR-122, miR-181a,miR-192, and miR-200b miRNAs were signicantly correlatedwith a
severity of NAFLD-specic liver pathomorphological features, with the strongest correlation occurring with miR-
34a. These observations suggest that the plasma levels of miRNAs may be used as biomarkers for noninvasive
monitoring the extent of NAFLD-associated liver injury and susceptibility to NAFLD.
Published by Elsevier Inc.
Introduction
The incidence of nonalcoholic fatty liver disease (NAFLD) is in-
creasing dramatically in the United States and developed countries
(Bellentani et al., 2010; Tiniakos et al., 2010). It is estimated that 25%
30% of adults in the United States have NAFLD (Day, 2011; Pascale
et al., 2010) and that NAFLD accounts for 39% of newly diagnosed
cases of chronic liver disease (Pascale et al., 2010). NAFLD is composed
of several related liver disorders ranging from uncomplicated steatosis
to nonalcoholic steatohepatitis (NASH). More importantly, NAFLD is
considered to be a major risk factor for the development of other
chronic liver diseases,such as brosis, cirrhosis, and hepatocellular car-
cinoma (Siegel and Zhu, 2009; Starley et al., 2010; Welzel et al., 2011).
The underlying molecular mechanisms involved in the etiology and
pathogenesis of NAFLD are only partially understood and pharmaco-
therapy options are limited (Cohen et al., 2001). Supportive treatment
strategies for NAFLD are focused on managing the metabolic syndrome
and chronic inammation in the liver; thus, early detection of the dis-
ease is critical for improved prognosis and prevention of more serious
liver illnesses. A recent report by Paie et al. in a small cohort of patients
with NAFLD demonstratedthat isolated steatosis does progress to NASH
accompanied by a worsening of the metabolic syndrome (Pais et al.,
2011), and suggested the crucial need for continued hepatic monitoring
in patients diagnosed with uncomplicated NAFLD. Unfortunately, at the
present time, a liver biopsy is the only method for the diagnosis and as-
sessment of disease progression (Sanyal et al., 2011).
Even though the incidence of NAFLD in the general population is
appreciable, it is estimated that only 10% of those diagnosed will pro-
gress to liver brosis or cirrhosis (Siegel and Zhu, 2009). In addition, the
incidence of NAFLD varies greatly among racial groups (Browning et al.,
2004) and NAFLD is a highly heritable trait (Schwimmer et al., 2011).
Toxicology and Applied Pharmacology 262 (2012) 5259
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase;
LDH, lactate dehydrogenase; miRNAs, microRNAs; NAFLD, nonalcoholic fatty liver dis-
ease; NASH, nonalcoholic steatohepatitis; qRT-PCR, quantitative reverse transcription
real-time PCR.
The views expressed in this manuscript do not necessarily representthose of the U.S.
Food and Drug Administration.
Corresponding author at: Division of Biochemical Toxicology, NCTR, 3900 NCTR
Rd., Jefferson, AR 72079, USA. Fax: +1 870 543 7720.
E-mail address: igor.pogribny@fda.hhs.gov (I.P. Pogribny).
0041-008X/$ see front matter. Published by Elsevier Inc.
doi:10.1016/j.taap.2012.04.018
Contents lists available at SciVerse ScienceDirect
Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/ytaap
Author's personal copy
A recent genome-wide association study (GWAS) of the subjects with
histopathologically-conrmed NAFLD identied several single nucleo-
tide polymorphisms as potential genetic modiers of the NAFLD activity
score, brosis, inammation, and serum alanine aminotransferase (ALT)
levels (Chalasani et al., 2010). Because the pathogenesis of NAFLD is
complex and involves dysregulation of several interdependent physio-
logical processes, including lipid metabolism, insulin resistance, im-
mune response, inamation, oxidative stress, and apoptosis (Larter
et al., 2010; Marra et al., 2008), additional studies in human populations
or animal models of the human population (Rusyn et al., 2010)are
needed to elucidate the genetic determinants of NAFLD.
Investigating the molecular basis of how genetic factors inuence
the susceptibility to NAFLD in humans is frequently impractical and
always very complex, whereas using relevant animal models may
substantially overcome many limitations of human-only studies. Several
studies have demonstrated an inter-strain variability in the suscepti-
bility of mice to NAFLD, NASH, and hepatocarcinogenesis induced by
either high-fat diet, or methionine- and choline-decient diet (Hill-
Baskin et al., 2010; Pogribny et al., 2009; Yamazaki et al., 2008). Each
of these dietary models has some advantages and limitations. For in-
stance, feeding high-energy diets compromises the metabolic status
and induces obesity but does not uniformly cause liver injury (Maher,
2011). In contrast, feeding either a methionine- and choline-decient
or choline-decient diet to rats or mice causes liver injury similar to
NAFLD but does not attain the compromised metabolic status observed
in patients.
Previous reports have convincingly demonstrated that dysregulation
of miRNA expression is an important early event in the pathogenesis of
NAFLD in humans (Cheung et al., 2008) and mice (Wang et al., 2009a).
However, the association between inter-individual differences in sus-
ceptibility to NAFLD and altered miRNA expression is largely unknown.
To explore further the potential mechanisms of inter-individual
differences in susceptibility and severity of NAFLD-related liver injury,
we fed a panel of seven genetically diverse inbred mouse strains that
are parental lines in the Collaborative Cross (Aylor et al., 2011)the
choline- and folate-decient (CFD) diet that consistently induces fat-
related liver injury resembling pathomorphological features of human
NAFLD (Maher, 2011). We determined the inter-strain variability in
severity of NAFLD induced by the CFD diet and its association with aber-
rations in miRNA expression. We also assessed whether or not circulat-
ing blood miRNAs may be used as potential biomarkers for noninvasive
evaluation of liver injury in NAFLD. Because miRNA expression in mouse
liver varies little among inbred strains (Gatti et al., 2011) the establish-
ment of circulatory miRNA biomarkers as noninvasivediagnostics of the
severity of NAFLD may ll a critical gap in clinical practice.
Materials and methods
Animals and experimental design. Male A/J, C57BL/6J, C3H/HeJ,
129S1/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ mice (6 weeks of age)
were obtained from the Jackson Laboratory (Bar Harbor, ME). These
strains were selected because they provide an excellent representation
of the broad genetic diversity and their genomes have been fully se-
quenced (Yang et al., 2011). The mice were housed in sterilized cages
in a temperature-controlled room (24 °C) with a 12 h light/dark cycle,
and given ad libitum access to puried water and NIH-31 pelleted diet.
At 8 weeks of age, mice from each strain were allocated randomly into
control and experimental groups. Mice in the experimental groups
were maintained on the CFD diet, a diet lacking choline and folic acid
(Diet #519541, choline and folate decient, iron supplemented, and L-
amino acid dened diet; Dyets, Inc., Bethlehem, PA) for 12 weeks.
Mice in the control groups received the same diet supplemented with
0.4% methionine, 0.3% choline bitartrate, and 2 mg/kg folic acid. Diets
were stored at 4 °C before use and given ad libitum with replacement
twice a week. Body weights of the mice were recorded weekly. Five ex-
perimental and ve control mice from each strain were euthanized by
exsanguination following deep isourane anesthesia 12 weeks after
diet initiation. Blood was collected into BD vacutainer EDTA containing
blood collection tubes (BD Biosciences, Franklin Lakes, NJ) by direct
puncture of the heart. Plasma was isolated by centrifugation at
3000 rpm for 10 min at 4 °C according to the manufacturer's instruc-
tions and stored at 80 °C. The livers were excised and a slice of the
median lobe was xed in neutral buffered formalin for 48 h for histo-
pathological examination. The remaining liverwas snap-frozen immedi-
ately in liquid nitrogen and stored at 80 °C for subsequent analyses.
All experimental procedures were reviewed and approved by the Na-
tional Center for Toxicological Research Animal Care and Use
Committee.
Biochemical analyses. Serum triglyceride, ALT, aspartate amino-
transferase (AST), lactate dehydrogenase (LDH), total cholesterol,
and glucose levels were measured using an ACE Alera® Clinical Chem-
istry System (Alfa Wassermann Inc., West Caldwell, NJ) according to the
manufacturer's protocol.
Tissue processing, histological analysis and criteria for pathology
assessment. After 48 h, a slice of the median lobe of the liver that
was xed in 10% neutral buffered formalin was trimmed, processed,
and embedded in inltrating media (Surgipath Formula R®, Leica Bio-
systems, Richmond, IL), sectioned at approximately 5 μm, mounted
on a glass slide, and stained with hematoxylin and eosin. The liver
sections were examined histopathologically for NAFLD-specic le-
sions, including cytoplasmic alteration, steatosis, hepatocellular de-
generation, inammation, hepatocellular karyocytomegaly, and oval
cell proliferation, and graded using a severity score system for each
of the morphological parameters as follows: grade 0, absent; grade
1, minimal; grade 2, mild; grade 3, moderate; and grade 4, severe
changes. Total liver pathology scores were calculated as the mean se-
verity for all of the NAFLD-specic lesions detected in the livers of the
mouse strains.
RNA extraction and miRNA expression analysis by quantitative reverse
transcription real-time PCR (qRT-PCR). Total RNA was extracted
from liver tissue using miRNAeasy Mini kits (Qiagen, Valencia, CA)
according to the manufacturer's instructions. Total RNA (100 ng) was
used for qRT-PCRs of miR-122, miR-192, miR-34a, miR-200b, miR-
221, and miR-181a using TaqMan miRNA assays (Applied Biosystems,
Foster City, CA), according to the manufacturer's instructions.
SnoRNA202 was used as an endogenous control. The relative amount
of each miRNA was measured using the 2
ΔΔCt
method (Schmittgen
and Livak, 2008). All qRT-PCR reactions were conducted in triplicate
and repeated twice.
Total plasma RNA, including miRNA, was isolated using QIAzol re-
agent (Qiagen) according to the manufacturer's instructions with minor
modications. In brief, 700 μl of QIAzol reagent containing 1.2 μgofcar-
rier MS2 RNA (Roche Diagnostics Corporation, Indianapolis, IN) was
added to 100 μl of each plasma sample. The sample was mixed in a
tube, spiked with 4 μlof0.5μM cel-miR-54 miRNA (Qiagen), and
140 μl of chloroform was added. After mixing vigorously for 15 s, the
sample was then centrifuged at 12,000 gfor 15 min. The upper aqueous
phase was carefully transferred to a new collection tube, and precipi-
tated with 2 volumes of isopropanol, centrifuged at 18,000 gfor 15 min,
and washed with 75% ethanol. After air drying, the RNA was dissolved
in 30 μl RNase-free water. The quality and quantity of the RNA was
evaluated using a NanoDrop 200c spectrophotometer (Thermo Fisher
Scientic, Waltham, MA). The efciency of small RNA isolation was
monitored by determining the amount of spiked miRNA recovered by
using TaqMan miRNA assays (Applied Biosystems). Total RNA (1.5 μl
per reaction) was used for qRT-PCRs of miR-122, miR-34a, miR-200b,
miR-192, miR-221, and miR-181a using TaqMan miRNA assays (Applied
Biosystems), according to the manufacturer's instructions. The relative
53V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 5259
Author's personal copy
amount of each miRNA was measured using the 2
ΔΔCt
method and
normalized to mmu-miR-16, a ubiquitous non-liver-specicmiRNA.
Statistical analyses. Results are presented as mean±S.D. Clinical
chemistry values, body weights, and miRNA levels were analyzed by
two-way analysis of variance (ANOVA), with pair-wise comparisons
being made by the StudentNewmanKeuls method. When necessary,
the data were natural log transformed before conducting the analyses
to maintain a more equal variance or normal data distribution. Histopa-
thology scores were evaluated by one-way ANOVA, using strain as the
xed factor. Pearson productmoment correlation coefcients were
used to determine the relationship between miRNA levels and histopa-
thology scores. P-values b0.05 were considered signicant.
Results
Inter-strain differences in liver pathology elicited by a choline- and
folate-decient diet
Over the course of this 12 week study, all mice fed the CFD diet
gained weight, with the 129S1/SvImJ mice gaining signicantly more
than the methyl-sufcient control mice, and the WSB/EiJ mice gaining
signicantly less than the methyl-sufcient control mice (Table 1).
These results are in contrast to some of the previous reports that found
a substantial loss of the body weight, often between 20 and 40%, as a re-
sult of feeding mice various formulations of lipogenic methyl-decient
diets, e.g., methionine- and choline-decient, or choline-decient diets
(Ariz et al., 2010; Hebbard and George, 2011; Larter and Yeh, 2008).
This is considered as one of the major disadvantages of the mouse
model of NAFLD induced by these diets (Ariz et al., 2010; Hebbard and
George, 2011; Larter and Yeh, 2008). A signicant increase, 1.53-fold,
in relative liver weight was observed in six of the seven strains fed the
CFD diet (Table 1).
Serum triglycerides, cholesterol, and glucose levels were affected
by the methyl donor-decient diet in a strain-dependent manner
(Table 1). A decrease in triglycerides was signicant in ve of the
seven strains tested and a greater than 50% reduction was observed
in CAST/EiJ mice. The magnitude of change in serum cholesterol and
glucose levels was less pronounced. Only two strains, PWK/PhJ, and
WSB/EiJ, exhibited a greater than 50% signicant reduction in choles-
terol and a signicant decrease in serum glucose concentrations was
found in WSB/EiJ mice only.
Feeding the CFD diet resulted in a signicant increase in the activity
of serum enzyme markersof liver injury, ALT and AST,in C57BL/6J, C3H/
HeJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ strains (Table 1),which was signif-
icantly correlated with the degree of liver injury(Supplementary Fig. 1).
Serum activity of LDH was increased signicantly in CAST/EiJ, PWK/PhJ,
and WSB/EiJ strains by 1.9, 2.4, and 2.6-fold, respectively.
Histopathological evaluation of liver injury revealed the presence of
NAFLD-associated pathomorphological features, including cytoplasmic
alteration, steatosis, necrosis, inammation, oval cell hyperplasia, and
karyomegaly in all mice fed the CFD diet (Table 2). Interestingly, the
magnitude of the liver injury varied greatly among strains with the ex-
tent of total liver injury increasing in the following order according to
the total liver pathology scores: A/JC57BL/6JC3H/ HeJ b129S1/
SvImJ CAST/EiJbPWK/PhJbWSB/EiJ. Lipid accumulation in hepato-
cytes was characterized by microvesicular, macrovesicular, and most
commonly mixed (microvesicular and macrovesicular) patterns of fat
deposition. Hepatocytes with microvesicularfat accumulation appeared
foamy, with the cytoplasm partially or completely lled with numer-
ous small lipid vacuoles, which did not displace the nucleus to the pe-
riphery. This pattern of steatosis was most pronounced in A/J mice
(Fig. 1C). Macrovesicular fatty change was morphologically character-
ized by hepatocytes mostly containing a moderately large to large and
well-dened single rounded vacuole within each cell.The nucleus and
cytoplasm were displaced to the periphery in such cells. This pattern
was commonly observed in strains exhibiting the greatest degree of
liver injury, such as WSB/EiJ mice (Fig. 1D).
Inammatory foci (Figs. 1C and D, marked by arrowheads) were
composed of a mixed population of polymorphonuclear and mononu-
clear cells, mainly neutrophils, macrophages, and lymphocytes. Single-
cell necrosis was one of the most common histopathological observa-
tions in both of the PWK/PhJ and WSB/EiJ mouse strains, but was most
severe in the WSB/EiJ mice. Karyomegaly, characterized as 24times
larger than typical hepatocyte nuclei, was found in CFD diet-fed mice
from PWK/PhJ and WSB/EiJ strains (Fig. 1; compare larger sized nuclei
in Figs. 1C and D). Hyperplasia of oval cells, stem cells of bile ductular
epithelium that appeared as single or double rows of oval- or round-
shaped cells organized in linear arrays in liver sinusoids, was also
observed in choline- and folate-decient PWK/PhJ and WSB/EiJ mice.
Effect of a choline- and folate-decient diet on hepatic microRNA
expression prole
Dysregulation of hepatic expression of miR-34a, miR-122, miR-
181a, miR-192, miR-200b, and miR-221 was previously reported to be
a common feature in human (Cheung et al., 2008)andmouseNAFLD
(Pogribny et al., 2010; Wang et al., 2009a). Because an inter-strain
Table 1
Inter-strain differences in pathology endpoints in mice fed the choline- and folate-decient diet.
Strain Group Body
weight
(g)
Change of
body weight
(g)
Relative liver
weight
(%)
ALT
(U/l)
AST
(U/l)
LDH
(U/l)
Triglycerides
(mg/dl)
Cholesterol
(mg/dl)
Glucose
(mg/dl)
A/J Control 25.4±1.51 6.62±1.26 4.16±0.25 162.8±85.0 203.2± 16.9 1180± 280 175.2 ± 11.7 94.0± 4.5 214.4 ±17.2
MCFD diet 23.2± 2.41 5.02± 1.02 4.76 ±0.45 118.4 ±7.7 204.8 ±32.3 1287±231 94.0±7.1* 116.4 ±15.7 194.8 ±16.5
C57BL/6J Control 30.8±1.22 11.88 ±1.02 4.17 ±0.22 49.6 ±8.3 135.6 ±26.6 1148± 475 188.0± 38.8 102.4 ±21.5 203.6 ± 39.2
MCFD diet 30.7± 2.98 11.90± 3.40 6.39± 0.95* 194.8± 50.6* 209.2± 36.11430 ±293 110.8 ±13.8114.8±14.1 274.4 ±31.3
C3H/HeJ Control 32.4±1.80 11.50±1.92 4.19± 0.14 57.6± 13.0 129.6 ±21.6 1149 ±438 166.4± 23.0 153.2± 4.8 197.6± 9.8
MCFD diet 34.8± 4.33 13.40± 3.40 7.44± 0.47* 266.4± 54.6* 259.2± 54.5* 1472 ±336 126.0 ±10.7* 149.6 ±8.9 202.0 ± 23.5
129S1/SvImJ Control 27.2±2.22 7.88±1.26 3.42±0.19 191.2±92.4 368.0± 99.1 1480± 235 171.2 ± 18.6 138.4± 7.6 248.0± 12.7
MCFD diet 27.7± 1.30 10.14± 1.21* 7.60± 2.05* 336.4 ±68.6 455.6± 50.4 2415 ±631 101.6 ±19.9* 122.6 ±11.6 219.0± 19.4
CAST/EiJ Control 16.9± 0.29 4.46± 0.51 4.16 ± 0.44 184.8±66.9 255.8± 65.4 1227± 198 233.2 ± 22.5 83.6± 5.9 278.8 ±25.9
MCFD diet 17.2± 0.23 4.75± 0.68 7.92 ±0.24* 375.5 ±60.1* 374.5 ±60.7 2316± 574* 104.5± 11.8* 57.0 ±24.3 329.0 ±36.5
PWK/PhJ Control 18.6± 1.27 2.86± 1.49 4.75 ±0.83 89.6± 28.2 197.2 ± 62.4 1066± 199 162.4 ±12.1 84.4 ±5.4 199.6±22.7
MCFD diet 20.1± 1.18 1.96± 0.73 13.2 ±0.72* 760.8 ±74.4* 576.0 ±66.0* 2529±313* 106.0 ±8.4* 33.4 ±8.4* 170.0 ±4.5
WSB/EiJ Control 21.1 ±0.80 6.84 ± 0.51 4.26 ± 0.05 103.6 ± 32.1 329.2 ± 73.5 1184± 343 108.4 ± 5.5 114.4 ± 3.0 195.6 ± 15.5
MCFD diet 19.3± 0.85 4.54± 0.577.60 ±0.34* 745.2 ±42.2* 617.6 ±71.0* 3090±301* 113.2 ± 11.7 30.0 ±3.0* 153.6± 6.4*
Bold Signicantly different from age-matched control mice;
*Signicantly different from age-matched control mice (p-value 0.01);
†—Signicantly different from age-matched control mice (p-value 0.05).
54 V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 5259
Author's personal copy
difference in liver injury phenotype elicited by the CFD diet was ob-
served in this study, we investigated whether or not hepatic expression
of these miRNAs may reect variability in the degree of NAFLD or may
be associated with strain-specic susceptibility to liver injury. The ex-
pression of miR-122 was down-regulated by 25 to 60% in the livers of
A/J, C57BL/6J, PWK/PhJ, and WSB/EiJ mice fed the CFD diet (Fig. 2). No
change was observed in C3H/HeJ, 129S1/SvlmJ, or CAST/EiJ mice. MiR-
192 was down-regulated by 50% only in C57BL/6J mice and slightly
up-regulated in CAST/EiJ mice.
The expression of miR-34a, miR-200b, and miR-181a was signi-
cantly increased in CFD diet-fed mice of a majority of strains in a
strain-dependent manner, with the maximum response occurring in
WSB/EiJ mice (Fig. 2). Up-regulation of hepatic expression of miR-221
was most pronounced (>10-fold) in PWK/PhJ mice, with a smaller,
but signicant, increases being observed in C3H/HeJ, 129S1/SvlmJ,
CAST/EiJ, and WSB/EiJ mice. In contrast, feeding the CFD diet did not af-
fect the expression of a ubiquitous non-liver-specic miRNAlet-7c in all
but WSB/EiJ mice (Supplementary Fig. 2).
Effect of a choline- and folate-decient diet on plasma microRNA
expression prole
Plasma levels of miRNAs have been shown to be correlated with
the degree of liver injury in response to drug treatment (Laterza et
al., 2009; Wang et al., 2009b). We evaluated the differences in pat-
terns of miRNA changes in plasma of mice fed control or CFD diet.
Table 2
Summary of the type and extent of hepatic lesions in mice fed the choline- and folate-decient diet for 12 weeks.
Strain Group Lesion
Karyomegaly Steatosis Necrosis, hepatocyte Inammation Oval cell hyperplasia Cytoplasmic alteration
A/J Control 0/5
a
0/5 0/5 1/5 (0.2±0.5)
b
0/5 5/5 (1.6 ±0.6)
MCFD diet 0/5 5/5 (1.4 ±0.6) 0/5 5/5 (1.0 ±0.0) 1/5 (0.2± 0.5) 4/5 (1.2±0.8)
C57BL/6J Control 0/5 0/5 1/5 (0.2±0.5) 1/5 (0.2 ±0.5) 0/5 2/5 (0.8 ±1.1)
MCFD diet 0/5 5/5 (1.8 ±0.5) 0/5 5/5 (1.4 ±0.6) 3/5 (0.6± 0.6) 3/5 (0.6±0.6)
C3H/HeJ Control 0/5 0/5 0/5 0/5 0/5 0/5
MCFD diet 0/5 5/5 (2.6 ±0.6) 0/5 5/5 (1.0 ±0.0) 0/5 0/5
129S1/SvImJ Control 0/5 0/5 0/5 1/5 (0.2± 0.5) 0/5 5/5 (1.8 ±0.5)
MCFD diet 0/5 5/5 (3.8 ±0.5) 0/5 5/5 (1.4 ±0.6) 1/5 (0.2 ±0.5) 0/5
CAST/EiJ Control 0/5 0/5 0/5 0/5 0/5 2/5 (0.8± 1.1)
MCFD diet 4/4 (1.0 ±0.0) 4/4 (3.5 ±0.6) 0/4 3/4 (0.8± 0.5) 1/4 (0.3 ±0.5) 3/4 (1.0±0.8)
PWK/PhJ Control 0/5 0/5 0/5 1/5 (0.2± 0.5) 0/5 1/5 (0.2 ±0.5)
MCFD diet 5/5 (1.4 ±0.6) 5/5 (3.8 ±0.5) 5/5 (1.0± 0.0) 5/5 (2.0± 0.0) 5/5 (1.2 ±0.5) 5/5 (4.0 ±0.0)
WSB/EiJ Control 0/5 0/5 0/5 1/5 (0.2±0.5) 0/5 4/5 (1.0±0.7)
MCFD diet 5/5 (2.0 ±0.0) 5/5 (4.0 ±0.0) 5/5 (3.8± 0.5) 5/5 (3.8± 0.5) 5/5 (3.6 ±0.6) 5/5 (2.2 ±0.5)
a
Lesion prevalence.
b
(Mean severity).
Fig. 1. Representative hematoxylin and eosin staining of liver tissues from control A/J and WSB/EiJ mice (A,B) mice fed the choline- and folate-decient diet (C,D).(A) Representa-
tive liver section from control A/J mice. (B) Representative liver section from control WSB/EiJ mice. (C) Representative liver section from A/J mice fed the CFD diet for 12 weeks.
Minimal steatosis and inammation (arrows) in the livers. (D) Representative liver section from WSB/EiJ mice fed the CFD for 12 weeks. Severe steatosis, inammation (arrow-
heads) and necrosis (arrows) of hepatocytes in the livers.
55V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 5259
Author's personal copy
Fig. 2. Expression changes of miR-122, miR-34a, miR-200b, miR-192, miR-221, and miR-181a miRNAs in the livers of control mice and mice fed the choline- and folate-decient
diet.The miRNA expression data are presented as fold change of each miRNA normalized to that of snoRNA202 in the livers of mice fed the CFD diet compared to control mice
(n=5, mean ±SED). Gray bars control groups; black bars CFD diet groups.* Signicantly different from age-matched control mice.
Fig. 3. Levels of miR-122, miR-34a, miR-200b, miR-192, miR-221, and miR-181a miRNAs in plasma of control mice and mice fed the choline- and folate-decient diet. The miRNA
expression data are presented as fold change of each miRNA normalized to that of normalized to mmu-miR-16, a ubiquitous non-liver-specic miRNA in plasma of mice fed the CFD
diet compared to control mice. (n =5, mean ±SED). Gray bars control groups; black bars CFD diet groups.* Signicantly different from age-matched control mice.
56 V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 5259
Author's personal copy
Fig. 3 demonstrates that there were strain-specic differences in the
levels of miRNAs in the mice fed the CFD diet. Notably, in A/J mice,
one of the strains with the lowest extent of methyl donor-decient
diet-induced liver injury, none of the miRNAs was increased by feeding
the CFD diet. In contrast, in plasma of PWK/PhJ and WSB/EiJ mice,
strains that exhibited the greatest degree of liver injury, each of miRNAs,
with the exception of miR-200b was signicantly increased by choline
and folate deciency.
Because strain-dependent effects of treatment on miRNA levels
in liver and plasma were observed, we tested whether the inter-
strain difference in the degree of liver injury correlated with miRNA
expression. Fig. 4 shows that the induction of miR-34a, miR-181a,
miR-200b and miR-221 in the livers is signicantly correlated with
the histopathology score in CFD diet-fed mice, with the strongest cor-
relations being observed with miR-181a and miR-200b. Interestingly,
expression of miR-122 and miR-192 in the livers, two of the most
abundant hepatic miRNAs (Gatti et al., 2011), did not correlate with
the extent of liver injury.
Fig. 5 shows that plasma levels of each of the miRNAs were signif-
icantly correlated with the degree of liver injury induced by the CFD
diet, with strongest correlation occurring with miR-34a, whereas
level of circulating miRNA let-7c did not differ (Supplementary
Fig. 2).
Discussion
The results of the present study demonstrate that feeding a mul-
tistrain panel of inbred mice the CFD diet for 12 weeks resulted in
strain-specic changes in biochemical indicators of liver injury in plasma
(Table 1) and accumulation of microscopically observed lesions in
the livers similar to human NAFLD (Table 2,Fig. 1). The previous
comprehensive studies have established that dietary-based models of
NAFLD induced by methionine- and choline-decient or choline-
decient diets are the best models to study mechanisms of liver injury
in NAFLD, despite the fact that some of the features do not resemble
pathogenesis of NAFLD in humans (Ariz et al., 2010; Hebbard and
George, 2011; Larter and Yeh, 2008; Maher, 2011). One of the major dis-
advantage of the mouse NAFLD model induced by methionine- and
choline-decient or choline-decient diets is associated with signicant
loss of body weight (Ariz et al., 2010; Hebbard and George, 2011; Larter
and Yeh, 2008). The results of the present study showing that feeding
the methionine-containing CFD diet induced liver injury without the
loss of body weight, overcome this shortcoming of the commonly used
methionine- and choline-decient or choline-decient diets.
The magnitude of biochemical indicators of liver injury in plasma
and the extent of histomorphological changes in the livers ranged,
with the order being: A/JC57BL/6JC3H/HeJb129S1/SvImJCAST/
EiJbPWK/PhJbWSB/EiJ. More importantly, the pathomorphological
alterations in the livers were accompanied by different expression pat-
terns of hepatic miR-122, miR-181a, miR-192, miR-34a, miR-200b, and
miR-221.
miRNA-122 is the most abundant and highly liver-specic miRNA,
accounting for 70% of all miRNAs in the adult liver (Girard et al., 2008;
Lewis and Jopling, 2010). The expression of hepatic miR-122 has been
reported to decrease signicantly in individuals with viral- and alcohol-
induced liver injuries and NASH (Cheung et al., 2008; Morita et al.,
2011) and to be inversely correlated with the severity of liver histopa-
thology. Similar down-regulation of miR-122 has been reported in ex-
perimental animals with NAFLD (Wang et al., 2009b; Pogribny et al.,
2010). However, the results of present study demonstrate clearly the
existence of inter-strain variability in expression of hepatic miR-122
in response to feeding the CFD diet. Specically, down-regulation of
Fig. 4. Correlation plots of total liver pathology scores and induction of miRNAs in the livers of mice fed the choline- and folate-decient diet.Total liver pathology scores represent
the mean severity for all of the lesions detected in the liver of mouse. Each symbol represents individual animal in the CFD diet group in respected mouse strain.
57V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 5259
Author's personal copy
miR-122 was found only in the livers of A/J, C57BL/6J, PWK/PhJ, and
WSB/EiJ mice only. More importantly, while changes in miR-122 ex-
pression in the livers may be reective of liver injury, the down-
regulation of this miRNA was not associated with severity of liver injury.
This was evidenced by the fact that changes in hepatic miR-122 expres-
sion did not correlate with the magnitude of pathomorphological
changes in the livers of choline- and folate-decient mice (Fig. 4). Like-
wise, the expression of another highly abundant and liver-specic
miRNAs, miR-192, was not associated with the severity of liver dam-
age induced by a CFD diet. In contrast, expression of miR-34a, miR-
200b, miR-221 and miR-181a miRNAs increased across strains, with
the greatest magnitude being found in sensitive PWK/PhJ, and WSB/
EiJ strains. More importantly, up-regulation of miR-34a, miR-181a,
miR-200b, and miR-221 in the livers of CFD diet-fed mice strongly
correlated with a severity of NAFLD-related pathomorphological
changes in the livers.
The most notable nding in this study is that plasma levels of each
of the miRNAs were signicantly correlated with the severity of liver
injury induced by the CFD diet, with strongest correlation occurring
with miR-34a.
It has been suggested that plasma miRNAs may be potential bio-
markers to diagnose and monitor diseases (Cortez and Calin, 2009), in-
cluding several types of liver pathologies, ranging from drug-induced
liver injury to chronic viral hepatitis and NAFLD (Wang et al., 2009b;
Laterza et al.,2009; Bihrer et al., 2011; Cermelli et al., 2011). Specically,
the increased levels of plasma miR-122 alone or the combination of
miR-122 and miR-192 have been used to detect liver injury induced
by traditional liver toxicants, including trichlorobromomethane, carbon
tetrachloride, and acetaminophen in rats and mice (Wang et al., 2009b;
Laterza et al., 2009). Importantly, several recent clinical studies have
demonstrated that the levels of circulating miR-122, miR-192, and
miR-34a are signicantly higher in patients with acetoaminophen-
induced acute liver injury, NAFLD, and chronic hepatitis C infection
suggesting that plasma miRNAs may be useful biomarkers to monitor on-
going damage of hepatocytes and the magnitude of necroinammation
(Cermelli et al., 2011; Starkey Lewis et al., 2011).Theresultsofthepresent
study showing a high and signicant correlation between the levels
of miR-122, miR-181a, miR-192, miR-34a, miR-200b, and miR-221 in
plasma and a severity of NAFLD-specic pathomorphological changes
in the livers of mice fed the CFD diet reinforce this suggestion. More
importantly, the lack of changes in the plasma level of ubiquitous
non-liver-specic miRNA let-7c indicates that these circulating miRNAs
may be potential liver-specic biomarkers for noninvasive evaluation of
liver injury in NAFLD. In addition, a strong correspondence between
correlation of the levels on miRNAs and the activity of ALT and AST in
plasma with the extent of liver injury suggests that miRNAs may serve
as independent robust biomarkers of liver injury (Fig. 5 and Supple-
mentary Fig. 1).
In conclusion, the results of our study demonstrate that develop-
ment of NAFLD in mice induced by a choline- and folate-decient
diet is associated with altered expression of hepatic miRNAs and
their subsequent elevation in plasma. More importantly, the severity
of NAFLD is strongly associated with levels of circulating miR-122,
miR-181a, miR-192, miR-34a, miR-200b, and miR-221 that mirror
the magnitude of NAFLD-associated liver injury. These results suggest
that circulating miRNAs may be applied in human population-based
studies as sensitive genetic background-independent indicators for
noninvasive monitoring the development and extent of NAFLD-
associated liver injury. Additionally, they can be used as potential
noninvasive indicators of susceptibility to NAFLD liver injury.
Supplementary data related to this article can be found online at
doi:10.1016/j.taap.2012.04.018.
Fig. 5. Correlation plots of total liver pathology scores and induction of miRNA levels in plasma of mice fed the choline- and folate-decient diet.Total liver pathology scores rep-
resent the mean severity for all of the lesions detected in the liver of mouse. Each symbol represents individual animal in the CFD diet group in respected mouse strain.
58 V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 5259
Author's personal copy
Conict of interest statement
The authors declare that there are no conicts of interest.
References
Ariz, U., Mato, J.M., Lu, S.C., Martines-Chantar, M.L., 2010. Nonalcoholic steatohepatitis,
animal models, and biomarkers: what is new? Methods Mol. Biol. 593, 109136.
Aylor, D.L., Valdar, W., Foulds-Mathes, W., Buus, R.J., Verdugo, R.A., Baric, R.S., Ferris,
M.T., Frelinger, J.A., Heise, M., Frieman, M.B., Gralinski, L.E., Bell, T.A., Didion, J.D.,
Hua, K., Nehrenberg, D.L., Powell, C.L., Steigerwalt, J., Xie, Y., Kelada, S.N.P.,
Collins, F.C., Yang, I.V., Schwartz, D.A., Branstetter, L.A., Chesler, E.J., Miller, D.R.,
Spence, J., Liu, E.Y., McMillan, L., Sarkar, A., Wang, J., Wang, W., Zhang, Q.,
Broman, K.W., Korstanje, R., Durrant, C., Mott, R., Iraqi, F.A., Pomp, D., Threadgill,
D., Pardo-Manuel de Villena, F., Churchill, G.A., 2011. Genetic analysis of complex
traits in the emerging collaborative cross. Genome Res. 21, 12131222.
Bellentani, S., Scaqlioni, F., Marino, M., Begogni, G., 2010. Epidemiology of non-
alcoholic fatty liver disease. Dig. Dis. 28, 155161.
Bihrer, V., Friedrich-Rust, M., Kronenberger, B., Forestier, N., Haupenthal, J., Shi, Y.,
Peveling-Oberhag, J., Radeke, H.H., Sarrazin, C., Herrmann, E., Zeuzem, S., Waidmann,
O., Piiper, A., 2011. Serum miR-122 as biomarker of necroinammation in patients
with chronic hepatitis C virus infection. Am. J. Gastroenterol. 106, 16631669.
Browning, J.D., Szczepaniak, L.S., Dobbins, R., Nuremberg, P., Horton, J.D., Cohen, J.C.,
Grundy, S.M., Hobbs, H.H., 2004. Prevalence of hepatic steatosis in an urban popu-
lation in the United States: impact of ethnicity. Hepatology 40, 13871395.
Cermelli, S., Ruggieri, A., Marrero, J.A., Ioannou, G.N., Beretta, L., 2011. Circulating
microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver dis-
ease. PLoS One 6, e23937.
Chalasani, N., Guo, X., Loomba, R., Goodarzi, M.O., Haritunians, T., Kwon, S., Cui, J.,
Taylor, K.D., Wilson, L., Cummings, O.W., Chen, Y.D., Rotter, J.I., Nonalcoholic
Steatohepatitis Clinical Research Network, 2010. Genome-wide association study
identies variants associated with histologic features of nonalcoholic fatty liver
disease. Gastroenterology 139, 15671576.
Cheung, O., Puri, P., Eicken, C., Contos, M.J., Mirshahi, F., Maher, J.W., Kellum, J.M., Min,
H., Luketic, V.A., Sanyal, A.J., 2008. Nonalcoholic steatohepatitis is associated with
altered hepatic MicroRNA expression. Hepatology 48, 18101820.
Cohen, J.C., Horton, J.D., Hobbs, H.H., 2001. Human fatty liver disease: old questions and
new insights. Science 332, 15191523.
Cortez, M.A., Calin, G.A., 2009. MicroRNA identication in plasma and serum: a new
tool to diagnose and monitor diseases. Expert Opin. Biol. Ther. 9, 703711.
Day, C.P., 2011. Non-alcoholic fatty liver disease: a massive problem. Clin. Med. 11, 176178.
Gatti, D.M., Lu, L., Williams, R.W., Sun, W., Wright, F.A., Threadgill, D.W., Rusyn, 2011.
MicroRNA expression in the livers of inbred mice. Mutat. Res. 714, 126133.
Girard, M., Jacquemin, E., Munnich, A., Lyonnet, S., Henrion-Caude, A., 2008. miR-122, a
paradigm for the role of microRNAs in the liver. J. Hepatol. 48, 648656.
Hebbard, L., George, J., 2011. Animal models of nonalcoholic fatty liver disease. Nat.
Rev. Gastroenterol. Hepatol. 8, 3544.
Hill-Baskin, A.E., Markiewski, M.M., Buchner, D.A., Shao, H., DeSantis, D., Hsiao, G.,
Subramaniam, S., Berger, N.A., Croniger, C., Lambris, J.D., Nadeau, J.H., 2010. Diet-
induced hepatocellular carcinoma in genetically predisposed mice. Hum. Mol.
Genet. 18, 29752988.
Larter, C.Z., Yeh, M.M., 2008. Animal models of NASH: getting both pathology and met-
abolic context right. J. Gastroenterol. Hepatol. 23, 16351648.
Larter, C.Z., Chitturi, S., Heydet, D., Farrel, G.C., 2010. A fresh look at NASH pathogenesis.
Part 1: the metabolic movers. J. Gastroenterol. Hepatol. 25, 672690.
Laterza, O.F., Lim, L., Garrett-Engele, P.W., Vlasakova, K., Muniappa, N., Tanaka, W.K.,
Johnson, J.M., Sina, J.F., Fare, T.L., Sistare, F.D., Glaab, W.E., 2009. Plasma microRNAs
as sensitive and specic biomarkers of tissue injury. Clin. Chem. 55, 19771983.
Lewis, A.P., Jopling, C.L., 2010. Regulation and biological function of the liver-specic
miR-122. Biochem. Soc. Trans. 38, 15531557.
Maher, J.J., 2011. New insights from rodent models of fatty liver disease. Antioxid.
Redox Signal. 15, 535550.
Marra, F., Gastaldelli, A., Svegliati Baroni, G., Tell, G., Tiribelli, C., 2008. Molecular basis
and mechanisms of progression of non-alcoholic steatohepatitis. Trends Mol. Med.
14, 7281.
Morita, K., Taketomi, A., Shirabe, K., Umeda, K., Kayashima, H., Ninomiya, M., Uchiyama,
H., Soejima, Y., Maehara, Y., 2011. Clinical signicance and potential of hepatic
microRNA-122 expression in hepatitis C. Liver Int. 31, 474484.
Pais, R., Pascale, A., Fedchuck, L., Charlotte, F., Poynard, T., Ratziu, V., 2011. Progression
from isolated steatosis to steatohepatitisand brosisin nonalcoholicfatty liver disease.
Clin. Res. Hepatol. Gastroenterol. 35, 2328.
Pascale, A., Pais, R.A., Ratziu, V., 2010. An overview of nonalcoholic steatohepatitis:
past, present and future directions. J. Gastrointestin. Liver Dis. 19, 415423.
Pogribny, I.P., Tryndyak, V.P., Bagnyukova, T.V., Melnyk, S., Montgomery, B., Ross, S.A.,
Latendresse, J.R., Rusyn, I., Beland, F.A., 2009. Hepatic epigenetic phenotype prede-
termines individual susceptibility to hepatic steatosis in mice fed a lipogenic
methyl-decient diet. J. Hepatol. 51, 176186.
Pogribny, I.P., Starlard-Davenport, A., Tryndyak, V.P., Han, T., Ross, S.A., Rusyn, I., Beland,
F.A., 2010. Difference in expression of hepatic microRNAs miR-29c, miR-34a, miR-
155, and miR-200b is associated with strain-specic susceptibility to dietary non-
alcoholic steatohepatitis in mice. Lab. Invest. 90, 14371446.
Rusyn, I., Gatti, D.M., Wiltshire, T., Kleeberger, S.R., Threadgill, D.W., 2010. Toxi-
cogenetics: population-based testing of drug and chemical safety in mouse models.
Pharmacogenomics 11, 11271136.
Sanyal, A.J., Brunt, E.M., Kleiner, D.E., Kowdley, K.V., Chalasani, N., Lavine, J.E., Ratziu, V.,
McCullough, A., 2011. Endpoints and clinical trial design for nonalcoholic
steatohepatitis. Hepatology 54, 344353.
Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative
C(T) method. Nat. Protoc. 3, 11011108.
Schwimmer, J.B., Celedon, M.A., Lavine, J.E., Salem, R., Campbell, N., Schork, N.J.,
Shiehmorteza, M., Yokoo, T., Chavez, A., Middleton, M.S., Sirlin, C.B., 2011. Herita-
bility of nonalcoholic fatty liver disease. Gastroenterology 136, 15851592.
Siegel, A.B., Zhu, A.X., 2009. Metabolic syndrome and hepatocellular carcinoma: two
growing epidemics with a potential link. Cancer 115, 56515661.
Starkey Lewis, P.J., Dear, J., Platt, V., Simpson, K.J., Craig, D.G., Antoine, D.J., French, N.S.,
Dhaun, N., Webb, D.J., Costello, E.M., Neoptolemos, J.P., Moggs, J., Goldring, C.E.,
Park, B.K., 2011. Circulating microRNAs as potential markers of human drug-
induced liver injury. Hepatology 54, 17671776.
Starley, B.Q., Calcagno, C.J., Harrison, S.A., 2010. Nonalcoholic fatty liver disease and
hepatocellular carcinoma: a weighty connection. Hepatology 51, 18201832.
Tiniakos, D.G., Vos, M.B., Brunt, E.M., 2010. Nonalcoholic fatty liver disease: pathology
and pathogenesis. Annu. Rev. Pathol. 5, 145171.
Wang, B., Majumder, S., Nuovo, G., Kutay, H., Volinia, S., Patel, T., Schmittgen, T.D.,
Croce, C., Ghoshal, K., Jacob, S.T., 2009a. Role of microRNA-155 at early stages of
hepatocarcinogenesis induced by choline-decient and amino acid-dened diet
in C57BL/6 mice. Hepatology 50, 11521161.
Wang, K., Zhang, S., Marzolf, B., Troisch, P., Brightman, A., Hu, Z., Hood, L.E., Galas, D.J.,
2009b. Circulating microRNAs, potential biomarkers for drug-induced liver injury.
Proc. Natl. Acad. Sci. U. S. A. 106, 44024407.
Welzel, T.M., Graubard, B.I., Zeuzem, S., El-Serag, H.B., Davila, J.A., McGlynn, K.A., 2011.
Metabolic syndrome increases the risk of primary liver cancer in the United States:
a study in the SEER-Medicare database. Hepatology 54, 463471.
Yamazaki, Y., Kakizaki, S., Takizawa, D., Ichikawa, T., Sato, K., Takagi, H., Mori, M., 2008. Inter-
strain differences in susceptibility to non-alcoholic steatohepatitis. J. Gastroenterol.
Hepatol. 23, 276282.
Yang, H., Wang, J.R., Didion, J.P., Buus, R.J., Bell, T.A., Welsh, C.E., Bonhomme, F., Yu, A.H.,
Nachman, M.W., Pialek, J., Tucker, P., Boursot, P., 2011. Subspecic origin and haplotype
diversity in the laboratory mouse. Nat. Genet. 43, 648655.
59V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 5259
    • The status of cytosine DNA and histone H3K4 trimethylation , a well-established major histone transcriptionactivating mark [21], in the normal livers of A/J mice, a strain characterized by a mild NAFLD-like liver injury, and WSB/EiJ mice, a strain that exhibits severe NASHlike liver injury induced by choline and folate deficiency [20] , was evaluated by methylated DNA immunoprecipitation (meDIP) and chromatin immunoprecipitation (ChIP) assays in combination with Agilent Mouse 2 × 105 K CpG Island Microarrays that cover 16,030 CpG islands of the mouse genome. Unsupervised hierarchical clustering of the CpG methylation and histone H3K4me3 data showed that A/J and WSB/EiJ mouse strains could be distinguished by their hepatic CpG island methylation and histone H3K4 methylation profiles (Fig. 1).
    [Show abstract] [Hide abstract] ABSTRACT: Background Nonalcoholic fatty liver disease (NAFLD) is a major health problem and a leading cause of chronic liver disease in the United States and Western countries. In humans, genetic factors greatly influence individual susceptibility to NAFLD; nonetheless, the effect of inter-individual differences in the normal liver epigenome with regard to the susceptibility to NAFLD has not been determined. Results In the present study, we investigated the association between the DNA methylation status in the livers of A/J and WSB/EiJ mice and the severity of NAFLD-associated liver injury. We demonstrate that A/J and WSB/EiJ mice, which are characterized by significant differences in the severity of liver injury induced by a choline- and folate-deficient (CFD) diet exhibit substantial differences in cytosine DNA methylation in their normal livers. Furthermore, feeding A/J and WSB/EiJ mice a CFD diet for 12 weeks resulted in different trends and changes in hepatic cytosine DNA methylation. Conclusion Our findings indicate a primary role of hepatic DNA methylation in the pathogenesis of NAFLD and suggest that individual variations in DNA methylation across the genome may be a factor determining and influencing the vulnerability to NAFLD. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2617-2) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • For example, miR-192, a common miRNA associated with liver in the literature, has a number of different potential origins including kidney, heart, and peripheral blood mononuclear cells (Fang et al., 2015; Jenkins et al., 2012; Yamamoto et al., 2012). Because of its putative correlation with inflammatory processes (Yamamoto et al., 2012 ), assessment of miR-192 (and other miRNAs), in combination with liver-specific miR-122 measurements, may delineate the type and severity of inflammation (or other adverse processes) linked to the liver pathology (Church et al., 2016; Tryndyak et al., 2012; Yamaura et al., 2012). APAP, alcohol, and Toll-like receptor 9 þ 4 ligand exposures have been shown in mice to induce high circulating levels of inflammation-linked miR-155, miR-125b, and miR-146a, in addition to miR-122 (Bala et al., 2012).
    [Show abstract] [Hide abstract] ABSTRACT: Biomarker measurements that reliably correlate with tissue injury and that can be measured within accessible biofluids offer benefits in terms of cost, time, and convenience when assessing chemical and drug-induced toxicity in model systems or human cohorts. MicroRNAs (miRNAs) have emerged in recent years as a promising new class of biomarker for monitoring toxicity. Recent enthusiasm for miRNA biomarker research has been fueled by evidence that certain miRNAs are cell-type specific and are released during injury, thus raising the possibility of using biofluid-based miRNAs as a “liquid biopsy” that may be obtained by sampling extracellular fluids. As biomarkers, miRNAs demonstrate improved stability as compared with many protein markers and sequences are largely conserved across species, simplifying analytical techniques. Recent efforts have sought to identify miRNAs that are released into accessible biofluids following xenobiotic exposure, using compounds that target specific organs. Whereas still early in the discovery phase, miRNA biomarkers will have an increasingly important role in the assessment of adverse effects of both environmental chemicals and pharmaceutical drugs. Here, we review the current findings of biofluid-based miRNAs, as well as highlight technical challenges in assessing toxicologic pathology using these biomarkers.
    Full-text · Article · Aug 2016
    • Our miRNA profiling study revealed that miR-122 expression is increased in plasma of ZDF rats during disease progression of T2D. miR-122 is considered to be a highly sensitive and specific biomarker in blood, reflecting hepatocyte injury, as increased levels of miR-122 were described in various liver diseases, such as non-alcoholic steatohepatitis (NASH) [42]. In NASH patients and in animal models, circulating miR-122 levels are elevated in serum, whereas its expression is decreased in liver tissue [43,44].
    [Show abstract] [Hide abstract] ABSTRACT: The aim of the present pilot study was the identification of micro-RNA changes over time during the development and progression of type 2 diabetes (T2D) in Zucker diabetic fatty rats (ZDF rats). T2D is a complex metabolic disorder that is characterized, inter alia, by progressive failure of pancreatic β cells to produce insulin, but also by functional or morphological modifications of others organ, such as liver, adipose tissue and the cardiovascular system. Micro-RNAs are a novel class of biomarkers that have the potential to represent biomarkers of disease progression. In this study, the onset and progression of diabetes was followed in ZDF rats from six weeks until 17 weeks of age. After an initial phase of hyperinsulinemia, the animals developed T2D and lost the capacity to produce sufficient insulin. Circulating miRNAs were measured from plasma samples at four time points: pre-diabetes (six weeks of age), hyperinsulinemia (eight weeks), β cell failure (11 weeks) and late-stage diabetes (17 weeks) using TaqMan miRNA arrays. Bioinformatic analysis revealed distinct changes of circulating miRNAs over time. Several miRNAs were found to be increased over the course of the disease progression, such as miR-122, miR-133, miR-210 and miR-375. The most significantly decreased miRNAs were miR-140, miR-151-3p, miR-185, miR-203, miR-434-3p and miR-450a. Some of the miRNAs have also been identified in type 2 diabetic patients recently and, therefore, may have the potential to be useful biomarkers for the disease progression of T2D and/or the treatment response for anti-diabetic medications.
    Full-text · Article · May 2016
    • As such, it may constitute an important player during NAFLD triggering and progression. In fact, mice fed with choline-and folate-deficient (CFD) diet for 12 weeks, resulting in a NASH-like phenotype, display a progressive increase of both liver and plasma miR-181a, paralleling the severity of NAFLD-specific liver pathomorphological features [75]. In liver cirrhosis patients, serum levels of miR-181b were found significantly elevated [136].
    [Show abstract] [Hide abstract] ABSTRACT: Obesity and metabolic syndrome are growing epidemics worldwide and greatly responsible for many liver diseases, including nonalcoholic fatty liver disease (NAFLD). NAFLD often progresses to cirrhosis, end-stage liver failure and hepatocellular carcinoma (HCC), the most common primary liver cancer and one of the leading causes for cancer-related deaths globally. Currently available tools for the diagnosis of NAFLD staging and progression towards HCC are largely invasive and of limited accuracy. In light of the need for more specific and sensitive noninvasive molecular markers, several studies have assessed the potential of circulating microRNAs (miRNAs) as biomarkers of liver injury and hepatocarcinogenesis. Indeed, extracellular miRNAs are very stable in the blood, can be easily quantitated and are differentially expressed in response to different pathophysiological conditions. Although standardization procedures and larger, independent studies are still necessary, miRNAs constitute promising, clinically-useful biomarkers for the NAFLD-HCC spectrum.
    Full-text · Article · Mar 2016
    • Biomarkers reflect molecular alterations that occur concomitant with changes in normal physiological pathways, thereby connecting toxic chemical exposure to the presence or risk of clinical disease (DeCaprio, 1997aDeCaprio, , 1997b). Many studies have reported that circulating miRNAs are potentially stable biomarkers in response to liver disease (Trebicka et al., 2013; Tryndyak et al., 2012). For instance, circulating miR-122-5p and miR-192-5p have been reported to be linked to drug-induced liver injury, while circulating miR-28-5p and miR-32-5p have been suggested to be associated with PFOA exposure (Yan et al., 2014).
    [Show abstract] [Hide abstract] ABSTRACT: Aflatoxin B1 (AFB1) is a well-known human hepatotoxicant and genotoxicant. Recent studies demonstrated that aberrant miRNA expression patterns were correlated with the cellular and genetic lesions induced by chemicals. To explore the role of miRNAs in AFB1-induced hepatotoxicity and genotoxicity, we examined alterations in miRNA expression patterns in F334 rat livers after exposure to 100 μg/kg or 200 μg/kg AFB1 for 28 days. Using high-throughput sequencing, we discovered that rno-miR-34a-5p, rno-miR-200b-3p, and rno-miR-429 were up-regulated and that rno-miR-130a-3p was down-regulated in liver tissue from rats that received 200 μg/kg of AFB1; this finding was validated by real-time PCR. AFB1 treatment resulted in the upregulation of rno-miR-34a-5p and rno-miR-200b-3p in the rat H-4-II-E cell line similar to our in vivo observations. Moreover, rno-miR-34a-5p was transcriptionally elevated via p53 activation after AFB1 exposure. Upregulation of rno-miR-34a-5p suppressed the expression of the cell cycle-related genes CCND1, CCNE2 and MET and led to cell cycle arrest in the G0-G1 phase. The CBMN assay indicated that inhibition of rno-miR-34a-5p and p53 expression aggravated the DNA damage induced by AFB1, which might be associated with shortening of the DNA damage repair period. Circulating miR-34a-5p in rat sera preceded a significant increase in ALT activity and other miRNAs in the 100 μg/kg AFB1 group. These observations demonstrated that rno-miR-34a-5p responded sensitively to AFB1 exposure and facilitated p53 repair of DNA damage by impacting the cell cycle. Thus, circulating rno-miR-34a-5p may be a sensitive indicator for the induction of hepatic genotoxicity by AFB1 in rats.
    Full-text · Article · Sep 2015
    • Genetic background has been indicated as one of the possible reasons for the ostensible lack of reproducibility in published data regarding circulating miRNAs as markers for breast cancer [57, 58]. Furthermore, it has been reported that a choline-and folate-deficient diet causing nonalcoholic fatty liver disease (NAFLD) determined a different extent of modulation of some miRNAs, including miR- 122, in both liver and plasma of divergent strains of mice [59]. These changes in circulating miRNAs based upon genetic variation and diet corroborates the observations in our chicken model.
    [Show abstract] [Hide abstract] ABSTRACT: Circulating extra-cellular microRNAs (miRNAs) have emerged as promising minimally invasive markers in human medicine. We evaluated miRNAs isolated from total plasma as biomarker candidates of a response to an abiotic stress (feed deprivation) in a livestock species. Two chicken lines selected for high (R+) and low (R-) residual feed intake were chosen as an experimental model because of their extreme divergence in feed intake and energy metabolism. Adult R+ and R- cocks were sampled after 16 hours of feed deprivation and again four hours after re-feeding. More than 292 million sequence reads were generated by small RNA-seq of total plasma RNA. A total of 649 mature miRNAs were identified; after quality filtering, 148 miRNAs were retained for further analyses. We identified 23 and 19 differentially abundant miRNAs between feeding conditions and between lines respectively, with only two miRNAs identified in both comparisons. We validated a panel of six differentially abundant miRNAs by RT-qPCR on a larger number of plasma samples and checked their response to feed deprivation in liver. Finally, we evaluated the conservation and tissue distribution of differentially abundant miRNAs in plasma across a variety of red jungle fowl tissues. We show that the chicken plasma miRNome reacts promptly to the alteration of the animal physiological condition driven by a feed deprivation stress. The plasma content of stress-responsive miRNAs is strongly influenced by the genetic background, with differences reflecting the phenotypic divergence acquired through long-term selection, as evidenced by the profiles of conserved miRNAs with a regulatory role in energy metabolism (gga-miR-204, gga-miR-let-7f-5p and gga-miR-122-5p). These results reinforce the emerging view in human medicine that even small genetic differences can have a considerable impact on the resolution of biomarker studies, and provide support for the emerging interest in miRNAs as potential novel and minimally invasive biomarkers for livestock species.
    Full-text · Article · Dec 2014
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