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Biochimica et Biophysica Acta 1820 (2012) 1092–1101
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
PFOS-induced hepatic steatosis, the mechanistic actions on β-oxidation and
lipid transport
H.T. Wan a, Y.G. Zhao a, X. Wei a, K.Y. Hui a, J.P. Giesy b, c, d, Chris K.C. Wong a,⁎
a
Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University, 200 Waterloo Road, Kowloon Tong, Hong Kong, China
Department of Veterinary Biomedical Sciences & Toxicological Center, University of Saskatchewan, Canada
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China
d
Zoology Department, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia
b
c
a r t i c l e
i n f o
Article history:
Received 1 November 2011
Received in revised form 15 March 2012
Accepted 16 March 2012
Available online 28 March 2012
Keywords:
Perfluorooctane sulfonate
Lipid metabolism
β-oxidation
Fatty acid uptake
Very-low density lipoprotein
Liver metabolism
a b s t r a c t
Background: Perfluorooctane sulfonate (PFOS) was produced by various industries and was widely used in diverse consumer products. Human sample analysis indicated PFOS contamination in body fluids. Animal studies
revealed that PFOS tends to accumulate in livers and is able to induce hepatomegaly. However the underlying
mechanism of PFOS-elicited hepatotoxicity has not yet been fully addressed. The objective of this study is to
identify the cellular target of PFOS and to reveal the mechanisms of PFOS-induced toxicity.
Methods: In this study, mature 8-week old male CD-1 mice were administered 0, 1, 5 or 10 mg/kg/day PFOS for
3, 7, 14 or 21 days. Histological analysis of liver sections, and biochemical/molecular analysis of biomarkers for
hepatic lipid metabolism were assessed.
Results: PFOS-induced steatosis was observed in a time- and dose-dependent manner. The gene expression levels
of fatty acid translocase (FAT/CD36) and lipoprotein lipase (Lpl) were significantly increased by 10 and/or 5 mg/
kg PFOS. Serum levels of very-low density lipoprotein were decreased by 14 days of PFOS exposure (p b 0.05).
The rate of mitochondrial β-oxidation was also found to be significantly reduced, leading to the restriction of
fatty acid oxidation for energy production.
Conclusion: Taken together, the disturbance of lipid metabolism leads to the accumulation of excessive fatty
acids and triglycerides in hepatocytes.
General significance: Since PFOS-elicited pathological manifestation resembles one of the most common human
liver diseases—nonalcoholic fatty liver disease, environmental exposure to PFOS may attribute to the disease
progression.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Perfluoroalkyl acids (PFAAs) are a family of synthetic fluorinated
hydrocarbons (C4–C14) with the charged functional moiety of carboxylate, sulfonate or phosphonate. Because of their unique hydrophobic and
oleophobic properties, they have been extensively used in various
industrial and consumer products, such as surfactants, emulsifiers,
fire-fighting foams, stain-resistant coatings for fabrics, leather, oilresistant coatings for paper products, waxes and polishes [1]. However
the carbon-fluoride bonds render these compounds to be nonbiodegradable, leading to their persistence in the environment and
lengthy serum elimination half-life in animals [2]. The degree of PFAA
accumulation in body appears to be dependent on the carbon chain
⁎ Corresponding author at: Croucher Institute for Environmental Sciences, Department of Biology, 200 Waterloo Road, Kowloon Tong, Hong Kong, China. Tel.: +852
3411 7053; fax: + 853 3411 5995.
E-mail addresses: wanhinting@gmail.com (H.T. Wan), 08467234@hkbu.edu.hk
(Y.G. Zhao), sissywei_wx@hotmail.com (X. Wei), yuiiuh@hotmail.com (K.Y. Hui),
ckcwong@hkbu.edu.hk (C.K.C. Wong).
0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2012.03.010
length and the functional moieties of the compounds [3,4]. One of the
family members of PFAAs, perfluorooctane sulfonate (PFOS), are globally present in the environment and are widely distributed in human
populations and wildlife [5–8]. Due to the potential adverse health effects of PFOS, its use in industrial production was phased out in most
countries in 2002, except in China where it's still manufactured and
used today. Currently PFOS is listed under Annex B of the Stockholm
Convention in 2009 as one of the nine new persistent organic pollutants
(POPs).
Considerable numbers of studies have related PFOS exposure to
the development of hepatotoxicity in animals [3,9–11]. Exposure to
PFOS has been reported to cause an increase of liver weight, peroxisome
proliferations, increased expression of genes involved in fatty acid
oxidation and a lesser serum cholesterol concentration in rodents
[9,11,12]. The phenotypic observations related to peroxisome proliferations are suggested to be the consequence of PFOS-induced peroxisome
proliferator-activated receptor-alpha (PPAR-α) activations, leading to
the disturbance of lipid metabolism [13]. However a previous study
on PPAR-α null mice demonstrated that the action of PFOS was independent of PPAR-α activation. PFOS is described as a weak PPAR-α
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H.T. Wan et al. / Biochimica et Biophysica Acta 1820 (2012) 1092–1101
agonist [14,15]. These evidences imply that the possible molecular
targets and the underlying mechanism of PFOS-elicited hepatotoxicity
have not yet been identified. In this study, molecular, biochemical and
physiological approaches were used to identify the molecular targets
and the metabolic fingerprints with the intention of dissecting the
mechanisms of PFOS-induced hepatomegaly in mice.
2. Materials and methods
1093
liver tissue was homogenized in chloroform: methanol (2:1) solution.
Sulphuric acid (1.2 ml of 0.05%) was added for phase separation. After
removal of the upper phase, chloroform with 0.5% Triton X-100
(1 ml) was added to the lower phase and the solution was then
dried under nitrogen at room temperature. The pellet was then rinsed
with 1 ml of chloroform and was dried under nitrogen before it was
reconstituted with 0.5 ml deionized water. After incubation for
30 min at 37 °C, the solution was used for enzymatic determination
of triglycerides (TGs) (Triglycerides assay kit, Cayman, USA).
2.1. Experimental animals and chemicals
2.4. RNA isolation and quantitative PCR
All experimental animals were housed and handled in accordance
with Guidelines and Regulations in Hong Kong Baptist University.
Male CD-1 mice (6–8 weeks old) were purchased from the Laboratory
Animal Service Centre (LASEC) of the Chinese University of Hong
Kong (Hong Kong, China). The entire study was conducted in quadruplicate with mice that were received in 4 separate batches. The animals
were acclimatized for one week before the experiments. The mice were
randomly divided into 4 groups (at least 4 individuals per group) and
the mice were housed in polypropylene cages with sterilized bedding.
The animals were maintained under controlled temperature (23.5 °C),
12 h light–dark cycle (06:00–18:00) and were fed with standard
foods (LabDiet, 5001 Rodents Diet) and water (in glass bottles) ad
libitum. The body weight of each mouse was determined by use of an
electronic balance (Shiamdzu, Tokyo, Japan). Perfluorooctane sulfonate
(PFOS, 98% purity, sigma Aldrich) was dissolved in DMSO (b0.4%) before mixing with corn oil. The exposed groups were administered by
oral gavage with 1, 5, or 10 mg/kg/day PFOS in corn oil for 3, 7, 14 or
21 days in every morning. The control group was given corn oil with
DMSO (b0.4%). Then the animals were killed by cervical dislocation in
the morning on the designated dates. Blood sample was collected by
cardiocenthesis and the blood serum was prepared by centrifugation
at 3000 g for 15 min. The serum samples were stored at −20 °C until
further analysis. Livers were weighted and parts of them were immediately fixed in 4% paraformaldehyde for histological assessment or
homogenized in TriReagent (MRC, Cincinnati, USA) for total RNA isolation, according to the manufacturer's protocol. The rest of the liver
samples were immediately frozen in liquid nitrogen and stored at
−80 °C until analysis. Adipose tissue from fat pad was collected for
total RNA isolation.
2.2. Histological examination of the mouse livers
Histological assessment was conducted on the liver samples fixed
in 4% paraformaldehyde solution (Sigma). Each fixed sample was
dehydrated in ethanol gradient solutions, xylene and was then
embedded in paraffin. The paraffin-embedded tissue was cut into
5 μm sections. The sections were stained with hematoxylin and
were observed under a light microscope (Motic, Canada).
2.3. Liver lipid content determination
The method for quantifying liver lipid content was conducted as
described in the previous studies [16,17]. Approximately 100 mg of
Total RNA with an A260/A280 ratio of between 1.8 and 1.9 was used
for quantitative real-time PCR analyses. Complementary DNA was
synthesized from 150 ng of total cellular RNA using the High Capacity
RNA-to-cDNA Master Mix (Applied Biosystems, Foster City, CA).
Gene-specific primers were designed from the published sequences
(Table 1). Real-time PCR was conducted with a program that consisted of 3 min at 95 °C followed by 40 cycles of 95 °C for 15 s, 56 °C
for 20 s and 72 °C for 30 s. Standards and cDNAs from samples were
quantified by use of the StepOne Real-time PCR system using SYBR
Green Master mix (Applied Biosystems). The folds of change in the
transcript level vs. control were calculated and all data were normalized to the transcript levels of mouse actin. Occurrences of primerdimers and secondary products were evaluated by the use of melting
curve analysis. Control amplifications were done either without RT or
without RNA. All glass- and plastic-ware were treated with diethyl
pyrocarbonate and autoclaved.
2.5. Western blot analysis
For western blotting, the liver sample was homogenized in sodium
dodecyl sulfate (SDS) lysis buffer (2% SDS and 25% glycerol in 125 mM
Tris/HCl (pH6.8)) and was subjected to electrophoresis in a 10%
polyacrylamide gel. The gel was blotted onto a PVDF membrane
(PerkinElmer Life Sciences). Western blotting was conducted using
rabbit polyclonal antibody for FAT/CD36 (1:250, Abcam, UK) or rabbit
polyclonal antibody for cytochrome P450 4A (1:250, Abcam, UK),
followed by an incubation with horseradish peroxidase-conjugated
goat anti-rabbit antibody (Bio-rad, CA, USA). Specific bands were visualized with chemiluminescent reagents (Western-lightening Plus,
PerkinElmer Life Sciences, USA). The blot was then washed in PBS0.5% Tween20 and re-probed with rabbit polyclonal antibody for βactin (Sigma, USA).
2.6. Serum HDL and LDL/VLDL assay
Concentrations of high-density lipoprotein (HDL), low-density
lipoprotein and very low-density lipoprotein (LDL/VLDL) in the blood
serum were determined by using the “HDL and LDL/VLDL” assay kit
(EnzyChromTM, BioAssay System, USA). Briefly a serum sample
(20 μl) was mixed with a precipitating reagent (20 μl), followed by
centrifugation at 9500 g for 5 min to precipitate LDL/VLDL fraction.
The supernatant was collected as the HDL fraction, while the pellet
Table 1
DNA sequences of primers used in the present study.
Primer
Forward
Reverse
Actin
Acadm (acadm)
ACOX1 (acox1)
Apolipoprotein B-100 (apob)
Carnitine palmitoyltransferase-1a1 (Cpt1a)
Cytochrome P450 4A14 (Cyp4a14)
FAT/CD36 (FAT/CD36)
Lipoprotein lipase (Lpl)
TCTACGAGGGCTATGCTCTCC
CAATGATGTGTGCTTACTGTGTGA
GGACCTTCACTTGGGCATGTT
ACCTACCTGATGGCTCTGATCC
CAAGATAGCTTGTGAAAAGCACCA
ATTGGTTATGGTTTGCTCCTGTTG
GCCAAGCTATTGCGACATGATTA
ATCAACTGGATGGAGGAGGAGT
TCTTTGATGTCACGCACGATTTC
CCCTTCTTCTCTGCTTTGGTCTTA
ATCTCCAGATTCCAGGCCGG
GCTGTTAACTGCGTGGCTCA
GGCTCAGACAGTACCTCCTTCA
TCATAGTGGAAGGCTGGAGTCA
ATCCGAACACAGCGTAGATAGAC
TTCTTATTGGTCAGACTTCCTGCT
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was reconstitued by PBS. A working reagent (60 μl) was added to each
well containing 50 μl of samples or standard. After incubation at room
temperature for 30 min, the absorbance was measured at 340 nm.
2.7. β-oxidation assay
The method for the analysis of the rate of β-oxidation was performed as previously described [18,19]. Briefly, approximately 200 mg
of liver sample was removed upon dissection and was added into an
ice-cold isolation buffer (220 mmol/l L-mannitol, 70 mmol/l sucrose,
2 mmol/l HEPES and 0.1 mmol/l EDTA (pH7.2)). After washing off the
blood, the sample was homogenized in 10 volumes of the ice-cold isolation buffer. Part of the homogenate was boiled at 100 °C for 20 min
and was used as a negative control. An aliquot of the liver homogenate
(150 μl) was added into a 2 ml volumetric flask that contained an incubation buffer (0.12 M KCl, 75 mMTris/HCl (pH 7.4), 10 mM potassium
phosphate, 5 mM magnesium chloride, 1 mM EDTA, 1 mM NAD +,
5 mM ATP, 5 mM ADP, 10 μM FAD, 100 μM Coenzyme A, 0.5 mM Lmalate and 0.5 mM L-carnitine). The substrate d 31-palmitic acid
(50 μM) (Isotec, Miamisburg, Ohio, USA) and an internal standard
tricosanoic acid (Sigma Aldrich, USA) were added to the flask. To measure peroxisomal β-oxidation, antimycin A (50 μmol/l) (Sigma Aldrich,
A
USA) and rotenone (10 μmol/l) (CalBiochem) were added to inhibit
mitochondrial β-oxidation. The flasks were then incubated for 45 min
at 37 °C in a shaking water bath. Incubation was ended by an addition
of 0.5 ml of 3 mmol/l perchloric acid (Sigma Aldrich, USA). After
10 min of 750 g centrifugation, the supernatant was loaded into a preconditioned Oasis HLB cartridge at the rate of 1 drop/sec, and was
then washed with 1 ml of 5% methanol. Each sample was eluted with
methanol/chloroform (5 ml, 1:1). The elute was then dried under nitrogen and was dissolved in 1 ml of 1:1 methanol/chloroform for the
determination of the remaining concentration of d 31-palmitic acid by
liquid chromatography (Agilent, Palo Alto, CA, USA) tandem mass spectrometry (AB/MDS Sciex 4000 Q TRAP). Data were calculated according
to the levels of d 31-palmitic acid consumed in the assays. Total βoxidation was determined by subtracting the boiled homogenate
(negative control) from the respective un-boiled homogenate.
Total β−oxidation ¼ Mitochondrial β−oxidation
þ Peroxisomal β−oxidation
Mitochondrial β-oxidation was then determined by subtracting
peroxisomal β-oxidation from the total β-oxidation.
Body Weight (g)
38
36
Ctrl (oil)
1 mg/kg
5 mg/kg
10 mg/kg
34
32
*
*
*
*
30
28
2
3
4
5
6
7
8
* p<0.05
9 10 11 12 13 14 15 16 17 18 19 20 21
Experimental Day
B
4
Absolute liver weight (g)
*
p < 0.001
#
# p < 0.01
3
p < 0.05
*
*
2
#
*
*
*
1
Ctrl
1 mg/kg
5 mg/kg
10mg/kg
0
Day 3
C
Day 7
Day 14
Day 21
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
Midclavicular length of liver (cm)
Day 21
2.5
p<0.003
c
2.0
1.5
a
ab
b
1.0
0.5
0.0
Ctrl
1 mg/kg
5 mg/kg 10 mg/kg
Fig. 1. Effects of PFOS exposure on mouse body and liver weights.Mice were administered 0, 1, 5, or 10 mg/kg/day PFOS by gavages in corn oil for 3, 7, 14 or 21 days. (A) The body
weights of PFOS-treated mice (10 mg/kg) were significantly reduced on the last 4 days of the exposure; however (B) the absolute liver weights were significantly increased in the
PFOS-exposed animals (⁎p b 0.001, #p b 0.01, p b 0.05 as compare to the control). (C) On day 21, the dissection showed the enlargement and the yellowish appearance in the liver of
the exposed animals. Significant increase in liver sizes was observed in 5 and 10 mg/kg PFOS-exposed group (p b 0.003).
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2.8. Statistical analysis
1095
effects. The analyses were conducted using SigmaStat for Windows,
version 3.5.
Statistical evaluation was conducted by the use of SPSS16. All data
were tested to be normally distributed and independent using the
Normal Plots in SPSS (Shapiro–Wilk significance was 0.05). Differences
between treatment groups and corresponding control groups were
tested for statistical significance by analysis of variance (ANOVA)
followed by Duncan's Multiple Range test (significance at p b 0.05)
SPSS16. Data are presented as the mean ± SEM. Statistical analyses of
linear regression data were analyzed by the Pearson product pairwise
comparisons to measure the strength of association between dose and
A
3. Results
3.1. Effects of PFOS exposure on body and liver weights
In mice exposed to 10 mg/kg PFOS, the gain in body weight was significantly less over the last 4 days of the exposure period (Fig. 1A). No
mice died during the study. Hepatomegaly was observed in mice
dosed with PFOS. The absolute liver weights were significantly induced
(i)
(ii)
Day 3
Ctrl
(iii)
(iv)
Day 14
Day 7
(v)
(vi)
Magnification: 400X
Day 21
C
Day 21
Ctrl
5 mg/kg
1 mg/kg
10 mg/kg
Total Liver Triglycerides (mg/g liver)
B
140
120
* p<0.001
*
# p<0.05
100
80
*
*
60
*
40
20
#
*
*
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
0
Day 3
Day 7
Day 14
Day 21
Fig. 2. Effects of PFOS exposure on the histological changes and triglyceride (TG) contents of the mouse livers. (A) Histological sections show an increase of cytoplasmic vacuolations in the
livers of the mice exposed to 10 mg/kg PFOS (i to v (200×)). The high magnification (400×) of hematoxylin staining of (vi) the liver sections from the 21 days of the exposure, showed
microvesicular (arrow heads) and macrovesicular steatosis (arrows). (B) Hematoxylin staining of the liver sections (200×) shows an increase in cytoplasmic vacuolations with different
doses of the exposure. (C) Liver triglyceride contents (mg) per gram liver were increased after PFOS exposure. (⁎p b 0.001, #p b 0.05, as compare to the control).
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H.T. Wan et al. / Biochimica et Biophysica Acta 1820 (2012) 1092–1101
by 10 mg/kg PFOS exposure after 3 days and 5 mg/kg PFOS after 7 days
onwards (Fig. 1B). The enlargement of liver and its yellowish appearance were first noted after 3 days of the exposure and were observed
in all the PFOS-exposed mice on day 21 (Fig. 1C, the left panel). The
liver sizes measured at midclavicular line were significantly increased
by 5 and 10 mg/kg PFOS as compared to the control (pb 0.003)
(Fig. 1C, the right panel). Statistical analysis was performed to analysis
the dose- and time-dependent effects of PFOS exposure on the absolute
liver weights. Linear regression analyses show significant positive correlations between the dose- or time- of the exposure to the increases
of absolute liver weights (Supplementary Fig. 1A).
5 mg/kg PFOS treatment group, a significant increase in Fat/CD36
transcript level was observed on day 21. A similar induction pattern
was noticed in the transcript levels of hepatic lipoprotein lipase
(Lpl) (Fig. 4B). In the adipose tissues, no noticeable difference in the
gene expression levels of the adipose FAT/CD36 and Lpl between the
control and the exposed groups were detected (Fig. 4C).
3.4. Effects of PFOS exposure on hepatic lipid export
The hepatic transcript levels of apolipoprotein B (apob) in the
mice exposed to 5 or 10 mg/kg PFOS for 14 or 21 days were significantly less than those of the controls (Fig. 5A). Noticeable decreases
in serum total LDL/VLDL levels were detected on days 14 and 21 of
the 10 mg/kg and/or 5 mg/kg at 21 days PFOS treatment (Fig. 5B).
Negative correlations between liver weights and total concentrations
of LDL/VLDL in blood serum were observed in the mice exposed to
PFOS for 14 or 21 days (Fig. 5C).
3.2. Histological and TG analyses of liver
A representative panel of histological staining of the liver sections
(400×) from the PFOS-exposed mice (10 mg/kg), illustrated an increase in the numbers and size of cytoplasmic vesicles (Fig. 2A). Macrovesicular steatosis was identified in the liver sections. Fig. 2B showed
the progressively increase in the number and the size of cytoplasmic
vacuolation in the liver cells (Fig. 2B).
The concentrations of triglyceride (TG) per gram of livers were
significantly greater in mice exposed to 10 mg/kg PFOS (Fig. 2C).
The concentrations of TG per gram of liver samples were positively
correlated with the increases of absolute liver weights for all experimental days as shown in the linear regression analysis (Fig. 3). All
PFOS-treated mice showed dose- and time-dependent effects on the increase of TG levels in the livers as indicated by linear regression analysis
(Supplementary Fig. 1B).
3.5. Effects of PFOS exposure on oxidation of fatty acids in liver
The transcript levels of cytochrome P450 4A14 (Cyp4a14) (Fig. 6A,
left panel), peroxisomal acyl-CoA oxidase 1 (acox1) (Fig. 6B, left
panel) and acyl-CoA dehydrogenase (acadm) (Fig. 6B, right panel)
were significantly higher in the mice exposed to 10 and/or 5 mg/kg
PFOS. The expression levels of the CYP4A catalyst protein were also
greater in mice exposed to PFOS than that of the controls (Fig. 6A,
right panel).
In the β-oxidation assays, the rate of enzymatic process can be calculated from the rate of consumption of d 31-palmitic acids. The rates
of both total and peroxisomal β-oxidation were significantly greater
in livers of the mice exposed to 5 or 10 mg/kg PFOS for 14 days
(Fig. 6C). However, the rate of mitochondrial β-oxidation was lesser
in all mice exposed to PFOS, as compared to the control mice.
3.3. Effects of PFOS exposure on the expression levels of fatty acid
translocase and lipoprotein lipase in liver and adipose tissues
The exposure of the mice to 10 mg/kg PFOS induced the mRNA
and protein expression levels of hepatic fatty acid translocase (FAT/
CD36) in both time- and dose-dependent manners (Fig. 4A). In the
Day 7
Triglyceride content (mg)/g
liver
Triglyceride content (mg)/g
liver
Day 3
40
35
y = 51.221x - 51.891
30
R² = 0.6619
25
20
15
10
5
0
0
0.5
1
1.5
Absolute liver weight (g)
70
60
y = 52.933x - 53.62
R² = 0.8784
50
40
30
20
10
0
0
2
0.5
1
p<0.001
Day 14
y = 100.29x - 102.14
R² = 0.9533
100
50
0
1
2
3
4
-50
Absolute liver weight (g)
p<0.001
Triglyceride content (mg)/g
liver
Triglyceride content (mg)/g
liver
200
0
2
2.5
p<0.001
Day 21
250
150
1.5
Absolute liver weight (g)
450
400
y = 165.92x - 238.75
350
R² = 0.9047
300
250
200
150
100
50
0
1
-50 0
3
4
Absolute liver weight (g)
2
p<0.001
Fig. 3. Time-dependent correlations between hepatic lipid contents and liver weights (g) of the PFOS-exposed mice. Positive correlations are observed on all experimental days
(p b 0.001).
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4. Discussion
1097
and the disturbance on hepatic lipid transport. The data reveal the
similar hallmark features as compared with the development of
NAFLD which is defined as significant lipid deposition (>5%) of liver
parenchyma, without history of excess alcohol consumption [20]).
Current evidence has revealed PFOS-induced hepatotoxicity, solely
on the basis of the observations on hepatomegaly, peroxisomes proliferations and hepatocellular vacuolations [9,11,12]. The underlying
mechanism and the clinical significance of PFOS-induced biochemical
changes in livers are not known. At present, our data illustrated the
time- and dose-dependent effects of PFOS exposure on hepatic lipid accumulation, resulting from the inhibition of mitochondrial β-oxidation
4.1. PFOS induce lipid accumulations in hepatocytes
In this study, PFOS exposure caused the reduction of body weight
but increased the absolute liver weight in the mice. The observations
Day 7
Day 3
A
FAT/CD36
β-Actin
35
FAT/CD36
p<0.003
25
β-Actin
*
20
5
*
Relative protein expression
level of FAT/CD36
mRNA expression level
of hepatic FAT/CD36
Day 21
Day 14
*
* p<0.001
30
15
10
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
5
0
Day 3
Day 7
Day 14
Day 21
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
4
*
*
*
*
*
Day 7
Day 14
* p < 0.001
3
*
2
1
0
Day 3
B
Day 21
mRNA expression level of hepatic Lpl
7
* p<0.001
*
6
5
4
*
3
*
2
1
0
Day 3
3.0
mRNA expression level
of Adipose FAT/CD36
2.5
2.0
1.5
1.0
0.5
0.0
Day 14
mRNa expression level of Adipose Lpl
C
Day 7
Day 3
Day 7
Day 14
Day 21
Day 21
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Day 3
Day 7
Day 14
Day 21
Fig. 4. PFOS exposure induced expression levels of genes involved in hepatic fatty acid uptake.Upon 10 mg/kg PFOS exposure on all experimental days, (A) both mRNA and protein
levels of fatty acid translocase (FAT/CD36) were significantly up-regulated. (B) The transcript levels of hepatic lipoprotein lipase (Lpl) were increased in the PFOS-exposed groups.
(C) The gene expression levels of adipose FAT/CD36 (left panel) and Lpl (right panel) were unchanged. (*p b 0.001, p b 0.003, as compare to the control).
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A
2.5
mRNA expression level of apob
* p < 0.001
2.0
p`<0.003
1.5
1.0
*
*
* *
0.5
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
0.0
Day 3
Serum total LDL/VLDL Level (mg/dl)
B
140
120
Day 14
Day 21
* p < 0.001
p<0.003
# p<0.05
100
#
80
60
*
40
20
0
C
Day 7
Day 3
Day 7
Day 14
Day 21
Day 14
Day 21
120
Serum LDL/VLDL level
(mg/dl)
Serum LDL/VLDL level
(mg/dl)
200
160
120
80
40
y = -35.441x + 173.78
R² = 0.6004
0
0.0
1.0
2.0
Liver weight (g)
3.0
4.0
100
80
60
40
y =-20.899x + 115.77
R² = 0.4278
20
0
0
1
p=0.002
2
Liver weight (g)
3
4
p=0.041
Fig. 5. PFOS exposure reduced expression levels on genes involved in hepatic lipid export.Upon 5 and 10 mg/kg PFOS exposure for 14 and 21 days, (A) the decrease of mRNA levels
of hepatic apolipoprotein B-100 (apob) and (B) serum total LDL/VLDL levels were noted (⁎p b 0.001, p b 0.003, #p b 0.05, as compare to the control). (C) Representative plots show
negative correlations between liver weights and serum total LDL/VLDL levels in PFOS-exposed groups.
are consistent with other reports [11,21]. Previous studies revealed the
loss of body weight was correlated with lesser circulating leptin levels,
resulting in the reduction of both appetite and food intake in rats
[21,22]. The increased liver weights in the PFOS-exposed mice were
associated with hepatocellular hypertrophy and the appearance of cellular vacuolations [9,14]. However the underlying mechanism leading
to these pathological consequences is not clear. In this study, we have
provided mechanistic data to reveal the time- and dose-dependent
effects of PFOS on hepatic lipid contents. Since the increase in the number and size of the cytoplasmic vacuolations in the liver was positively
correlated with the increase of hepatic lipid contents, it is reasonable
to assume that the vacuoles contained lipid. Therefore the histological
analysis suggested the unevenly distributed microvesicular steatosis
and mixed microvesicular/macrovesicular steatosis. The net retention
of lipids within hepatocytes is known to be the prerequisite factors for
NAFLD development, of which they are mostly in the form of triglycerides (TGs) [23,24]. The mixed steatosis is also a common histological
feature found in NAFLD patients [24–26]. Nevertheless the PFOS-
elicited reduction of body weight but an accumulation of liver TG may
represent a mal-utilization of lipids for energy production in the
exposed animals.
4.2. Fatty acid oxidation
In hepatic fatty acid metabolism, the very long-chain fatty acids
undergo cytochrome P450 enzyme-mediated ω-hydroxylation to form
toxic dicarboxylic acids and free radicals [27]. The dicarboxylic acids
then enter the peroxisomal and mitochondrial β-oxidation for chainshortening and ATP production. Previous studies suggested the upregulation of ω-hydroxylation and β-oxidation enzymes (Cyp4A, acox1
and acadm) and the mitochondrial membrane carnitine-dependent
enzyme shuttle (Cpt1a) upon PFOS exposure in rodents [4,9,28]. Our
data revealed similar results except the induction of Cpt1a (Supplementary Fig. 2). The up-regulation of the enzymes was suggested to be contributed by the activation of PPAR-α, which transcriptionally activates
the three important fatty acid oxidative processes in rodents [29,30].
Author's personal copy
H.T. Wan et al. / Biochimica et Biophysica Acta 1820 (2012) 1092–1101
1099
Day 7
Day 3
A
CYP 4A
β -Actin
250
CYP 4A
β -Actin
150
10
100
*
*
50
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
*
*
Day 3
Relative protein expression
level of CYP4A14
mRNA expression level
of adipose Cyp4a14
*
200
0
Day 7
Day 14
Day 21
8
3.0
**
6
*
4
*
*
Day 3
Day 7
*
2
Day 14
Day 21
5
* p < 0.001
#
*
#
#
# p < 0.05
2.5
2.0
1.5
1.0
0.5
0.0
* p < 0.001
mRNA expression level of acadm
mRNA expression level of acox1
3.5
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
* p < 0.001
0
B
Day 21
Day 14
* p < 0.001
#
3
Day 7
C
Day 14
*
#
*
2
1
0
Day 3
#
# p < 0.05
4
Day 3
Day 21
Day 7
Day 14
Day 21
35
# p < 0.05
Amount of d31-Palmitic
acid consumed (µM)
30
Day 14
p < 0.01
25
#
20
15
10
Total Beta-oxidation
Peroxisomal beta-oxidation
Mitochondrial Beta-oxidation
#
5
0
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
Fig. 6. PFOS exposure altered the rate of fatty acid oxidation.Upon 10 mg/kg PFOS exposure for 7, 14 and 21 days, the levels of (A) CYP4A14 mRNA (left panel) and protein (right
panel) as well as (B) acox1 mRNA (left panel) and acadm mRNA (right panel) were significantly up-regulated. (C) PFOS exposure for 14 days decreased the rate of mitochondrial βoxidation but increased the rate of total and peroxisomal β-oxidation (⁎p b 0.001, p b 0.01, #p b 0.05, as compare to the control).
In the present study, the primary mechanistic effect of PFOS was identified to be on the inhibition of mitochondrial β-oxidation, leading to the
accumulation of fatty acids (FAs) and so lipid in livers. Since FAs are the
natural PPAR ligands, one possible interpretation of the observation is
that the intra-hepatic FAs accumulation might activate PPAR-α and
the downstream target genes to alter the hepatic lipid metabolism.
We assumed that the activation of the PPAR-α is via FAs accumulation,
which was the secondary effect of PFOS intoxication. Nevertheless the
Author's personal copy
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H.T. Wan et al. / Biochimica et Biophysica Acta 1820 (2012) 1092–1101
activation of the ω- and peroxisome β-oxidation would produce longchain fatty acyl-CoA, which were needed to be catabolized in mitochondria. In the PFOS-treated mouse, the rate of mitochondrial β-oxidation
was suppressed by 5 and 10 mg/kg PFOS exposure as compare to the
control. While mitochondrial β-oxidation could not cope with the
increase of ω- and peroxisomal β-oxidation, this would lead to further
accumulation of excess fatty acids and production of oxidative stress
[31,32]. This positive feedback loop would cause much disturbance on
hepatic lipid homeostasis.
4.3. Hepatic fatty acid uptake
Hepatic fatty acid uptake is governed by the availability of plasma
free fatty acids (FFAs) and the ability of hepatocytes to uptake FFAs
[33,34]. The capacity of FFAs trafficking to liver is an important factor
contributing to TG accumulation. The trafficking depends on the
activities of the hepatic lipoprotein lipase (Lpl) and fatty acid translocase (FAT/CD36) [34]. The hepatic Lpl hydrolyses lipoproteins whereas FAT/CD36 transports the FFAs from plasma to the liver. In this
study, the significant inductions of hepatic FAT/CD36 and Lpl were
detected upon 10 mg/kg PFOS treatment. The observation implies
an increase of hepatic fatty acid uptake in the PFOS-exposed mice.
Too high concentrations of free fatty acids accumulated in the liver
might prompt to the de novo formation of TGs. Similar observation
has been reported in human NAFLD cases and the abnormally elevated
hepatic FAT/CD36 activity likely relate to the pathology of steatosis
[34].
4.4. Hepatic export of lipids
Another process that contributes to liver TG accumulation is the
hepatic exports of TGs. The secretion of VLDL is a key step in the
prevention of lipid accumulation in the liver [23,34]. Hepatic
VLDL formation relies on the incorporation of lipoproteins with
apolipoprotein-B100 (apob). Our data indicated that 10 mg/kg PFOS
caused significant reduction in the gene expression levels of apob.
This may lead to the reduction in the formation of VLDL in the liver.
Consistently, serum analysis also revealed the significant reduction
in LDL/VLDL levels after 21 days treatment of high dose PFOS. It
resulted in the decrease of VLDL and TG export in the PFOS-exposed
mice. Intriguingly in human NAFLD, the reduction of TG removal
from liver through VLDL secretion predisposes the development of
steatosis [31,35].
5. Conclusion
The current hypothesis on the development of NAFLD in human is
thought to follow a “two hit pathways”, the initiation of liver steatosis
sensitizes liver fibrosis and inflammation [32]. Mitochondria dysfunction and the elevation of oxidative stress could promote the development to steatohepatitis. In addition, fat-induced insulin resistance is
strongly associated with human NAFLD. Currently there is no sufficient
evidence to indicate the association of PPAR-α activation with pathogenesis of human NAFLD. This claim is based on the discrepancy in
the effectiveness of using PPAR-α agonist for therapeutic treatment of
NAFLD in humans and rodents [36,37]. This discrepancy is probably
attributed by the very low expression levels of PPAR-α in human livers,
and the difference in the respective PPAR-α downstream targets
between rodents and humans [38]. In study upon which we report
here, the primary mechanistic effect of PFOS is on mitochondria βoxidation. The PFOS-elicited inhibition of mitochondria dysfunction
and the accumulation of hepatic TG are relevant to the initiation and
progression of liver steatosis in human NAFLD. The possible role of
PFOS in the development of NAFLD warrants further investigation.
Acknowledgements
This work is supported by the Collaborative Research Fund (HKBU 1/
CRF/08), University Grants Committee (CKC Wong). The research was
supported, in part, by a Discovery Grant from the National Science and
Engineering Research Council of Canada (Project # 326415-07). Prof.
JP Giesy was supported by the Canada Research Chair program, an at
large Chair Professorship at the Department of Biology and Chemistry
and State Key Laboratory in Marine Pollution, City University of Hong
Kong, The Einstein Professor Program of the Chinese Academy of
Sciences and the Visiting Professor Program of King Saud University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.bbagen.2012.03.010.
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Supplementary Fig.1
(A) Dose and time-dependent effect of PFOS on absolute liver weight
Day 3
Day 7
Absolute liver weights (g)
3
Day 14
R2= 0.936
(p<0.001)
4
R2= 0.944
(p<0.001)
3.5
Day 21
2.5
R2= 0.797
(p<0.001)
2
1.5
R2 =
0.254
(p=0.047)
Absolute liver weights (g)
3.5
1
0.5
Ctrl
1 mg/kg
R2= 0.88
(p<0.001)
5 mg/kg
3
10 mg/kg
2.5
R2= 0.834
(p<0.001)
2
R2= 0.463
(p=0.002)
1.5
R2= 0.129
1
0
2.5
5
7.5
10
0.5
12.5
0
5
10
15
20
25
Treatment Day
PFOS (mg/kg)
(B) Dose and time-dependent effect of PFOS on liver TG contents
140
Day 3
Day 14
R2= 0.879
(p<0.001)
Day 21
100
80
R2= 0.965
(p<0.001)
60
40
R2= 0.903
(p<0.001)
20
R2= 0.539
(p<0.001)
0
0
Ctrl
1 mg/kg
Day 7
120
2.5
5
7.5
PFOS (mg/kg)
10
12.5
Total triglycerides (mg)/ g liver
Total triglycerides (mg) / g liver
140
120
5 mg/kg
10 mg/kg
R2= 0.927
(p<0.001)
100
80
60
R2= 0.854
(p<0.001)
40
R2= 0.919
(p<0.001)
20
R2=0.518
0
0
5
10
15
Treatment Day
20
25
Relative protein expression level of C
Supplementary
Figure.2
8
6
4
(A)
0
mRNA expression level of CPT1a
2.5
2
Day 3
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
2.0
1.5
Day 7
Day 14
Day 21
1.0
0.5
0.0
Day 7
Ctrl
1 mg/kg
5 mg/kg
10 mg/kg
Day 14
Day 21