TOXICOLOGICAL SCIENCES 106(1), 162–171 (2008)
doi:10.1093/toxsci/kfn166
Advance Access publication August 18, 2008
Activation of Mouse and Human Peroxisome ProliferatorActivated
Receptor Alpha by Perfluoroalkyl Acids of Different Functional Groups
and Chain Lengths
Cynthia J. Wolf,1 Margy L. Takacs, Judith E. Schmid, Christopher Lau, and Barbara D. Abbott
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina 27711
Received May 22, 2008; accepted August 6, 2008
Perfluoroalkyl acids (PFAAs) are surfactants used in consumer
products and persist in the environment. Some PFAAs elicit adverse
effects on rodent development and survival. PFAAs can activate
peroxisome proliferator2activated receptor alpha (PPARa) and
may act via PPARa to produce some of their effects. This study
evaluated the ability of numerous PFAAs to induce mouse and
human PPARa activity in a transiently transfected COS-1 cell
assay. COS-1 cells were transfected with either a mouse or human
PPARa receptor-luciferase reporter plasmid. After 24 h, cells were
exposed to either negative controls (water or dimethyl sulfoxide,
0.1%); positive control (WY-14643, PPARa agonist); perfluorooctanoic acid or perfluorononanoic acid at 0.52100mM; perfluorobutanoic acid, perfluorohexanoic acid, perfluorohexane sulfonate, or
perfluorodecanoic acid (PFDA) at 52100mM; or perfluorobutane
sulfonate or perfluorooctane sulfonate at 12250mM. After 24 h of
exposure, luciferase activity from the plasmid was measured. Each
PFAA activated both mouse and human PPARa in a concentrationdependent fashion, except PFDA with human PPARa. Activation of
PPARa by PFAA carboxylates was positively correlated with carbon
chain length, up to C9. PPARa activity was higher in response to
carboxylates compared to sulfonates. Activation of mouse PPARa
was generally higher compared to that of human PPARa. We
conclude that, in general, (1) PFAAs of increasing carbon backbone
chain lengths induce increasing activity of the mouse and human
PPARa with a few exceptions, (2) PFAA carboxylates are stronger
activators of mouse and human PPARa than PFAA sulfonates, and
(3) in most cases, the mouse PPARa appears to be more sensitive to
PFAAs than the human PPARa in this model.
Key Words: perfluoroalkyl acids; PFAA; peroxisome
proliferatoractivated receptor alpha; PPARa; PFOS; PFOA;
PFNA; PFBA; transient transfection assay; COS-1 cells.
Disclaimer: This article has been reviewed by the National Health and
Environmental Effects Research Laboratory, U.S. Environmental Protection
Agency (U.S. EPA). The use of trade names is for identification only and does
not constitute endorsement by the U.S. EPA.
1
To whom correspondence should be addressed at Reproductive Toxicology
Division, National Health and Environmental Effects Research Laboratory,
Office of Research and Development, U.S. Environmental Protection Agency,
MD-67, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: wolf.
[email protected].
Published by Oxford University Press 2008.
Perflouroalkyl acids (PFAAs) are synthetic chemicals with
a carbon backbone saturated with fluorine and a charged
moiety on one end, making them both lipophobic and
hydrophobic, and very stable. These properties lend them to
wide consumer and industrial applications as surfactants, flame
retardants, water repellants, and oil repellants on food
packaging (Kissa, 2001; Renner, 2001). These properties also
make them persistent in the environment. PFAAs have been
found globally, in air, water, soil, and house dust (Boulanger
et al., 2005; Emmett et al., 2006; Giesy et al., 2001; Hansen
et al., 2002; Harada et al., 2006; So et al., 2004; Shoeib et al.,
2005). PFAAs have also been found in liver, fat, and serum of
wildlife everywhere from the polar regions to industrialized
areas (DeSilva and Mabury, 2006; Giesy and Kannan, 2001;
Giesy et al., 2001; Taniyasu et al., 2003), in human serum and
blood around the world (Butenhoff et al., 2004; Calafat et al.,
2006, 2007; Kannan et al., 2004; Kuklenyik et al., 2004; Olsen
et al., 2003; 2007b), in breast milk (Karrman et al., 2007;
Kuklenyik et al., 2004; Volkel et al., 2008), and in umbilical
cord blood (Apelberg et al., 2007). These PFAAs include not
only the well-studied perfluorooctanoic acid (PFOA) and
perfluorooctane sulfonate (PFOS) but also others such as
perfluorononanoic acid (PFNA), perfluorodecanoic acid
(PFDA), perfluorohexane sulfonate (PFHxS), perfluorobutanoic acid (PFBA), and perfluorobutane sulfonate (PFBS).
These chemicals are slow to clear from the body of most
animals, with a half-life estimated at days or months, and as
long as years in humans for PFOA, PFOS, and PFHxS (Olsen
et al., 2005; 2007a).
The presence and persistence of PFAAs, especially in human
body fluids, have drawn much attention and raised concern
about this class of compounds. This concern is heightened by
findings of toxic effects of PFAAs in experimental animals.
PFOS exposure in the rat and nonhuman primate was found to
reduce body weight and survival, increase liver weight, and
induce adenomas of the liver and thyroid (3M Company, 2003;
Seacat et al., 2002, 2003). PFOA exposure has a similar profile
of reduced body weight and liver toxicity and, in addition,
PFAAs ACTIVATE THE MOUSE AND HUMAN PPARa
induces adenomas of the Leydig cells and pancreatic acinar
cells in rodents (Biegel et al., 2001; Cook et al., 1992;
Goldenthal, 1978). Other PFAAs (perfluorohexanoic acid
[PFHxA], perfluoroheptanoic acid, and PFNA) were found to
induce hepatomegaly as well (Kudo et al., 2006). Developmental effects of PFOA and PFOS include full litter
resorptions, low birth weight, neonatal mortality, delayed eye
opening, and stunted mammary gland development in the
offspring of mice, rats, and/or rabbits (Case et al., 2001; Lau
et al., 2003; 2006; Luebker et al., 2005; Thibodeaux et al.,
2003; White et al., 2007). Recently, levels of PFOS and PFOA
in human cord blood were linked to low birth weights in infants
(Apelberg et al., 2007), although in another study (Fei et al.,
2007), there was no association between maternal PFAA levels
and low birth weight.
One mechanism of action by which PFAA induces these
effects may include activation of peroxisome proliferator
activated receptor (PPAR) a. PPARs are a class of ligandactivated transcription factors of the steroid/thyroid nuclear
hormone receptor superfamily (Dreyer et al., 1992). They are
involved in many cell processes including energy metabolism,
cell differentiation, and lipid homeostasis and are expressed in
many organs and species, as early as the developing embryo
(Auboeuf et al., 1997; Braissant and Wahli, 1998; Keller et al.,
2000; Peraza et al., 2006). There are three isoforms of PPAR,
a, b/d, and c. PPARa is primarily involved in lipid
homeostasis, fatty acid catabolism, peroxisome proliferation,
and inflammation (Escher and Wahli, 2000; Gonzalez et al.,
1998). It has been postulated that PFAAs can induce
hepatomegaly and liver tumors in rodents by activating
PPARa. PPARa induction of peroxisome proliferation is
associated with nongenetic hepatocarcinogenesis (Klaunig
et al., 2003). PFAAs are capable of activating PPARa in vitro
(Maloney and Waxman, 1999; Shipley et al., 2004; Vanden
Heuvel et al., 2006), inducing peroxisomal enzymes in male
rats (PFOA, PFNA, PFDA; Kudo et al., 2000), inhibiting
peroxisomal beta-oxidation in female rats (PFDA; Borges
et al., 1993), and inducing peroxisome proliferation in rats and
mice (Berthiaume and Wallace, 2002; Ikeda et al., 1985; 3M
Company, 2003). Consistent with PPARa-mediated responses,
PFOA is immunosuppressive in the mouse (Yang et al., 2000)
and anti-inflammatory in the rat (Griesbacher et al., 2008). In
addition, deletion of PPARa in knockout mice prevented the
postnatal lethality produced by gestational PFOA exposure
(Abbott et al., 2007). It is unclear whether peroxisome
proliferation is a mechanism of toxicity in humans. The
involvement of PPARa in inducing other toxic effects in
humans remains to be determined.
Transiently transfected cell models using COS-1 and 3T3L1 cells have been used to determine the ability of PFAAs to
bind PPARa (Bility et al., 2004; Intrasuksri et al., 1998;
Maloney and Waxman, 1999; Shipley et al., 2004; Takacs
and Abbott, 2007; Vanden Heuvel et al., 2006). These
PPARa-transfected cell models have been used to compare
163
relative activity in response to PFAAs. Takacs and Abbott
(2007) used COS-1 cells transiently transfected with
a PPARa, b/d, or c plasmid to evaluate the potential for
PFOA and PFOS to activate the mouse and human PPAR
isoforms. In the current study, we extend the investigation to
evaluate the ability of PFAAs with different carbon chain
lengths, including perfluoroalkyl carboxylates (PFBA,
PFHxA, PFOA, PFNA, and PFDA) and sulfonates (PFBS,
PFHxS, and PFOS) to activate the mouse and human PPAR
isoforms most likely involved in mediating some toxic effects
of PFAAs, the PPARa.
MATERIALS AND METHODS
Chemicals. WY-14643 (4-chloro-6-(2,3-xylidine)-pyrimidinylthioacetic
acid) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich
(St Louis, MO). PFOS (potassium salt; purity > 98%), PFHxA (purity > 97%),
and PFOA (ammonium salt; purity > 98%) were purchased from Fluka
Chemical (Steinheim, Switzerland). PFNA (purity 97%) and PFDA (purity
98%) were purchased from Aldrich (St Louis, MO). PFBA (ammonium salt;
28.9% solution in distilled water), PFBS (potassium salt; purity 98.2%, linearity
99.98%), and PFHxS (potassium salt; purity 98.6%) were a gift from 3M
Company (St Paul, MN). WY-14643 was dissolved in DMSO to make a 25mM
stock solution. PFAAs were dissolved in deionized distilled water (Picopure
Hydro Services and Supplies, Inc., Durham, NC) to make dilutions, except
PFDA, which was dissolved in DMSO. PFOS, poorly soluble, was dissolved in
boiling water to make stock and cooled to room temperature before adding to
dose solution. Chemical solutions were prepared fresh on the day of treatment.
Plasmids. The origin, preparation, and use of mouse and human PPARa
plasmids (a gift from Dr Jeffrey M. Peters and Dr John P. Vanden Heuvel, Penn
State University, PA) are described previously in Takacs and Abbott (2007).
Briefly, the plasmids contain a construct of the ligand-binding domain (LBD)
of mouse or human PPARa fused to the DNA-binding domain of Gal4 under
the control of an SV-40 promoter and a construct of an UAS-firefly luciferase
reporter under the control of a Gal4 DNA response element.
Cell culture and transactivation assay. Cell culture methods were
performed as previously described (Takacs and Abbott, 2007). Briefly, COS-1
cells (American Type Culture Collection, Manassas, VA) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY)
with 10% fetal bovine serum (Gibco) and antibiotic (0.2 mg/ml streptomycin
and 200 U/ml penicillin; Gibco). Cells were plated at a density of 104 cells per
100 ll DMEM per well in 96-well plates. After 24 h, cells were transfected
with the plasmid (1 lg/ll) in 10-ll serum-free DMEM using FuGENE 6
transfection reagent (Roche Diagnostics Corporation, Indianapolis, IN).
Transfection solutions were prepared in triplicate, one tube per plate, so that
each plate was a separate assay. DMEM containing serum (100 ll) was added
after 3 h of incubation with the transfection reagents. The medium was
aspirated and replaced with 100-ll serum-free DMEM containing test
chemicals 24 h after transfection. Cells were treated with WY-14643 (positive
control; 10lM), DMSO (control vehicle for WY-14643; 0.1%), deionized
distilled water (negative control vehicle for the PFAAs; 0.1%), or PFAA in the
following concentrations: PFOA or PFNA at 0.5100lM; PFBA, PFDA,
PFHxA, or PFHxS at 5100lM; or PFBS or PFOS at 1250lM. Solutions of
the PFAAs were prepared fresh on the day of treatment. Each compound was
tested in four or eight wells per concentration per plate in three plates per assay
in at least two assays to provide 2484 replicates for each concentration of
each compound. Twenty-four hours after treatment, cells were rinsed with
Dulbecco’s phosphate-buffered saline, lysed with reporter lysis buffer (cat#
E3971 or E1531; Promega, Madison, WI), and luciferase activity was measured
in relative luciferase units (RLUs) within an hour using the Luciferase reporter
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WOLF ET AL.
assay kit (Promega) on the LUMIstar Galaxy luminometer (BMG Labtechnologies, Durham, NC).
Viability and transfection control assays. Tests for cytotoxicity were
performed as described previously in Takacs and Abbott (2007). Briefly,
a fluorescence detection CellTiter-Blue cell viability kit (Promega) was used to
test the cell viability for each compound at each concentration in one plate per
compound. Transfection efficiency for the PPARa plasmids using the secreted
alkaline phosphatase concentration assay was described and tested previously
in our laboratory (Takacs and Abbott, 2007).
Statistics and calculations. Data were analyzed using SAS for Windows
V9.1. All data values were log10 transformed before analysis. Outliers were
defined by calculating residuals within a fixed effects linear model (SAS Proc
GLM) that accounted for dose group and plate, and residuals greater than 2.6
(roughly equivalent to using a two-sided p value of 0.01) were identified as
outliers. The data with outliers removed were analyzed by compound and
species with mixed effects linear models (SAS Proc Mixed) using restricted
maximum likelihood estimation, with dose group as a fixed effect and
experiment and plate (nested within experiment) as random effects. Differences
between each dose group and the control group were tested using Dunnett’s
test, and this was used to identify the lowest observed effects concentration
(LOEC). The responses across the dose range were tested with regression
analyses, providing slope and intercept estimates for each compound by
species. Goodness of fit for each model was estimated by calculating an R2
value for each model using SAS type III sums of squares. The difference in
response between species for each compound was tested as the difference in
slope between mouse and human, using a similar regression model that allowed
a separate intercept and slope for each species.
To allow direct comparison of responses between PFAA compounds,
a measurement was devised called the C20max, described below. Responses
were set on the same scale by defining the highest log-transformed RLU
obtained in all assays (1.11 RLU produced by PFDA in the mouse plasmid) as
the overall maximal possible response. The range of responses was set from
0 to 1.0. Thus, if 1.11 RLU ¼ 1.0 (or 100% response), then 20% of the
maximal response would be 0.22 RLU. With this relative response scale and the
regression formula for each slope, it is possible to calculate a C20max or
concentration at which each PFAA compound is predicted to produce 20% of
the overall maximal response. For example, shown here mathematically and
illustrated in Figure 1 for PFBA, a linear fit of the slope gives the regression
formula Y ¼ aX þ b, in which we set Y at 20% of maximal RLU or 0.22, a ¼
the slope (0.0043 for mouse PFBA in this example), and b ¼ the intercept
which is set to 0. Thus, 0.22 ¼ 0.0043X þ 0 and X (the C20max) ¼ 51lM.
RESULTS
The positive control, WY-14643, its negative vehicle control
(DMSO), and a negative vehicle control for the PFAAs were
run concurrently on every plate along with each PFAA
compound tested. PFAAs were tested at nine different
concentrations per plate. The positive control elicited a significant increase in activity over its vehicle control for every
assay, at p < 0.0001, indicating proper performance of the
assay. Except PFDA when tested with the human PPARa
plasmid, each PFAA elicited a significant dose-dependent
response compared to the vehicle control, indicating activation
of each PPARa by the PFAA. PFDA did not activate the
human PPARa. The lowest concentration of the PFAA that
produced an effect, or the LOEC, and the next lower
concentration tested which did not produce an effect, or the
no observable effects concentration (NOEC), are shown in
FIG. 1. Diagram showing the extrapolation of the C20max, or the
concentration at which a PFAA produces 20% of the overall maximal
response, using the slope of its regression line of the dose-response curve. Here,
the regression lines for human and mouse PPARa activity induced by PFBA
are shown. Maximal response value (highest log RLU obtained across the
entire set of experiments) was determined to be 1.11027, and 20% of this was
calculated to be 0.222055. This value was identified on the y-axis, and
a horizontal line was drawn, intercepting the linear dose-response regression
lines, to locate the concentrations on the x-axis for each species, which is the
20% maximal response concentration, or C20max. This graphical representation
illustrates the use of the regression formula (Y ¼ aX þ b) to estimate the
C20max for each compound and species.
Table 1. The NOEC and the LOEC were obtained by
comparing the luciferase activity produced by the PFAA at
each concentration to the activity in the vehicle control.
A lower LOEC or ppm indicates that a smaller concentration of
the PFAA is able to elicit a significant response. The longer
carbon chain carboxylates, PFOA and PFNA (mouse and
human) and PFDA (mouse), produced effects at low ppm
concentrations and were more active in the assay than the
shorter chain carboxylates or the sulfonates.
Overall response across concentrations was examined for
each compound, and a curve was fit to the data that best
characterized the dose-response. The dose-response for most
compounds was linear, although PFOA and PFNA produced
a biphasic curve that was linear in the 030lM range and
plateaued at higher concentrations. A generalized comparison
of the responsiveness of PPARa to the PFAAs can be made by
plotting these curves together (Fig. 2). In general, the
sulfonates were not as active as the carboxylates in either
species, and the carboxylates were more active in the mouse
PPARa than in the human. In Figure 3, the data are plotted by
compound and species. This figure illustrates that, for most
PFAA, the compounds elicited higher PPARa activity with the
mouse plasmid compared to the human. The slope of the
response to PFHxS was not different between the two species,
and with PFBS, activity was higher with the human plasmid
compared to the mouse. The slopes, p values, and r2 of the slopes
are shown in Table 2, along with the statistical comparison
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PFAAs ACTIVATE THE MOUSE AND HUMAN PPARa
Table 1
Summary of NOEC and LOEC for PFAA Transactivation of Mouse and Human PPARa in Transiently Transfected COS-1 Cells
LOEC
Compound
Species
Dose range (lM)
NOEC (lM)
(lM)
ppm (lg/ml)
p Value
PFBA (C4)
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
5–100
5–100
5–100
5–100
0.5–100
0.5–100
0.5–100
0.5–100
5–100
5–100
1–250
1–250
5–100
5–100
1–250
1–250
30
30
10
5
0.5
5
1
1
<5
100
120
20
10
5
60
20
40
40
20
10
1
10
5
5
5
> 100
150
30
20
10
90
30
9.24
9.24
6.28
3.14
0.43
4.3
2.3
2.3
2.6
> 51
50.7
10.1
8.76
4.38
48.4
16.15
< 0.0001
< 0.05
< 0.0001
< 0.05
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.05
ns
<0.01
< 0.0001
< 0.05
< 0.001
< 0.0001
< 0.05
PFHxA (C6)
PFOA (C8)
PFNA (C9)
PFDA (C10)
PFBS (C4)
PFHxS (C6)
PFOS (C8)
Note. ns, not significant.
between mouse and human. Except in the case of PFDA, which
was inactive with the human plasmid, p values of the regression
were significant, indicating a significant dose-response for these
compounds in each species.
In order to more directly compare responses, the outcomes
were adjusted to the same scale using a percentage of the maximal
response. The concentration of the PFAA that produces 20% of
the maximal response was extrapolated from the regression
FIG. 2. Plots of the dose-response activity of mouse (A, C) and human (B, D) PPARa induced by perfluoroalkyl carboxylates (A, B) and sulfonates (C, D) in
COS-1 cells. PFAA were prepared in serum-free DMEM. COS-1 cells were transfected with PPARa plasmid and exposed for 24 h to one PFAA per 96-well plate
at concentrations shown, with four or eight replicate wells per concentration, with three plates per assay in at least two assays. Data points are means of all assays
per PFAA compound per species. Dashed lines represent responses with the mouse plasmid; solid lines represent responses with the human plasmid. A key is
provided with the figure showing the identity of each compound.
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WOLF ET AL.
FIG. 3. Linear regression plots showing the dose-response activity of mouse and human PPARa induced by various PFAA compounds, shown by compound
and in order of increasing carbon chain length for the perfluoroalkyl acids in (A), (C), (E), (G), and (H), and for the sulfonates in (B), (D), and (F). PPARa plasmid
of mouse or human was transiently transfected into COS-1 cells, and cells were exposed to each PFAA in serum-free DMEM for 24 h at concentrations shown, in
four or eight replicate wells per concentration, in three replicate plates per assay, and in at least two assays. Dashed lines and open symbols represent mouse
plasmid and solid lines and solid symbols represent human plasmid. m, mouse; h, human.
formula and slope for each analysis and is referred to as the C20max
(Table 3, Fig. 1). PFNA (C9) and PFOA (C8) were the most
potent, eliciting significant PPARa activity at the lowest
concentrations of all the PFAAs (mouse, 5 and 6lM, respectively;
human, 11 and 16lM, respectively) followed by PFDA in mouse.
Activity of the plasmids decreased with decreasing chain length
of the carboxylate. Sulfonates were found to elicit activity in
either species at higher concentrations than the carboxylates.
PFBS, the sulfonate with the shortest chain length, induced the
least activity of any of the PFAAs tested.
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PFAAs ACTIVATE THE MOUSE AND HUMAN PPARa
Table 2
Regression Analysis of Dose-Response Activation by PFAA of Transfected Human and Mouse PPARa in COS-1 Cells
Mouse
Compound
PFBA
PFHxA
PFOAa
PFNAa
PFDA
PFBS
PFHxS
PFOS
Slope of regression line
Human
Regression p value
<
<
<
<
<
<
<
<
0.0043
0.0059
0.0354
0.0411
0.0111
0.0007
0.0029
0.0024
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
R2
Slope of regression line
0.71
0.48
0.85
0.82
0.87
0.81
0.84
0.80
0.0029
0.0048
0.0138
0.0207
0.00002
0.0011
0.0027
0.0008
Mouse versus human
Regression p value
<
<
<
<
0.0001
0.0001
0.0001
0.0001
ns
< 0.0001
< 0.0001
< 0.0001
R2
0.63
0.80
0.90
0.72
0.51
0.71
0.80
0.82
Slope difference p value
<
<
<
<
<
<
0.0001
0.01
0.0001
0.0001
0.0001
0.001
0.4334
< 0.0001
Note. ns, not significant.
For concentrations 020lM.
a
DISCUSSION
This study was undertaken based on the finding that PFOA
and PFOS can activate PPARa in vitro (Intrasuksri et al., 1998;
Maloney and Waxman, 1999; Takacs and Abbott, 2007; Vanden
Heuvel et al., 2006) and affect PPARa-mediated responses
(Berthiaume and Wallace, 2002; Ikeda et al., 1985; Kudo et al.,
2000; Wilson et al., 1995). It is important to know whether other
PFAA activate PPARa as well to explore the possible role of the
receptor in their mechanism of action as well. This study is the
first to examine the ability of multiple PFAAs (PFBA, PFBS,
PFHxA, PFHxS, PFOA, PFOS, PFNA, and PFDA) as a class to
activate PPARa in one transiently transfected COS-1 cell model.
This study demonstrates that many perfluoroalkyl carboxylates
and sulfonates activate the mouse and human PPARa in a transiently transfected COS-1 cell model and that the magnitude of
Table 3
Relative Responses of PPARa to PFAAs in Transiently
Transfected COS-1 cells, Measured by C20max
C20max (lM)
Compound
PFNA
PFOA
PFDA
PFHxA
PFBA
PFHxS
PFOS
PFBS
Mouse
Human
5
6
20
38
51
76
94
317a
11
16
na
47
75
81
262a
206
Note. na, not active; compounds are arranged in order of decreasing potency.
C20max, predicted concentration at which compound elicits 20% of the overall
maximal response.
a
Value exceeds highest concentration actually tested (250lM).
the response largely depends on the chain length of the carbon
backbone and the functional group.
In general, activation of PPARa increased with increasing
chain length of the carboxylate, up to C9. In the mouse PPARa,
the LOEC for the four-carbon (C4) PFBA was 40lM, indicating
a relatively weak agonist. The LOEC for PFHxA (C6) was
20lM, and the longer chained PFOA (C8), PFNA (C9) and
PFDA (C10) had LOECs ranging from 1 to 5lM, indicating
stronger agonist activity. Likewise, in the human PPARa, the
LOEC for PFBA was 40lM, the LOEC for PFHxA and PFOA
was 10lM, and the LOEC for PFNA was 5lM. Unexpectedly,
PFDA was inactive. This comparison is more discernable when
examining the data on a relative scale using the C20max values for
these PFAAs. PFBA had the lowest relative activity, indicated
by the highest C20max within species (51lM, mouse; 75lM,
human), and PFNA had the highest relative activity, indicated
by the lowest C20max (5lM, mouse; 11lM, human). This effect
of chain length on activation of PPARa is consistent with reports
of increased hepatomegaly and peroxisomal beta-oxidation by
PFAA with increasing chain length (Kudo et al., 2006) or
increased PPARa activity with increasing chain length of
phthalate compounds, also PPARa ligands (Bility et al., 2004;
Lampen et al., 2003). The association between PFAA chain
length and receptor activity may be a characteristic of the
PPARa LBD. Interestingly, in the current study, the PFAA acid
with the longest carbon chain length, PFDA, did not induce
activity from the human PPARa at any concentration. This may
indicate a difference in the size or conformation of the binding
site of the mouse and human PPARa LBD. The relatively large
molecule of the C10 compound may be too large and make it
stearically impractical to properly bind to the site of the human
PPARa LBD in its closed, active conformation, leading to little
or no activity, whereas C10 may tightly bind the mouse LBD
due to sequence differences at the site that allows a larger
conformation and activate the receptor.
The present study also demonstrated that the sulfonates, C4,
C6, and C8, induced lower activity in both mouse and human
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WOLF ET AL.
PPARa compared to the carboxylates. This confirms and adds
to our previous report that PFOS elicited lower activity than
PFOA (Takacs and Abbott, 2007) and is consistent with reports
from other laboratories (Maloney and Waxman, 1999; Shipley
et al., 2004; Vanden Heuvel et al., 2006).
In addition, the activation response of the mouse PPARa
was higher than that of the human for all PFAAs at each
concentration when compared by regression analysis across the
dose-response, with two exceptions; PFHxS induced identical
responses in each species, and PFBS induced higher activation
of human PPARa than of mouse. Slopes of the dose-response
curves with mouse PPARa were higher and roughly twice that
obtained with human PPARa in many cases (PFNA, PFBA,
and PFOA). Higher sensitivity in the mouse PPARa compared
to the human PPARa in response to other PPARa ligands
(Bility et al., 2004) or to PFOA or PFOS (Maloney and
Waxman, 1999; Takacs and Abbott, 2007; Vanden Heuvel
et al., 2006) has been demonstrated previously. The sequence
of LBD of the PPARa differs between mouse and human, and
it has been postulated that this difference and the differential
gene expression between human and rodent PPARa may be
responsible for the difference in response to PFAAs or other
ligands in the assay (Bility et al., 2004).
The present study confirms work by others who have shown
activation of mouse and human PPARa by PFOS (Shipley
et al., 2004) and PFOA (Intrasuksri et al., 1998; Maloney and
Waxman, 1999; Vanden Heuvel et al., 2006). Previous studies
and ours report similar LOECs and concentration ranges for
activation of PPARa by PFOA and PFOS. In COS-1 cells,
Maloney and Waxman (1999) reported activation of mouse and
human PPARa by PFOA beginning at concentrations ranging
from 0.5 to 1lM, and in 3T3-L1 cells, Vanden Heuvel et al.
(2006) reported activation by PFOA beginning at 50lM for
both mouse and human. PFOS was reported to activate mouse
PPARa in COS-1 cells at 8lM (Shipley et al., 2004). In our
study, the LOEC of PFOA were 1 and 10lM, and for PFOS,
the LOEC were 90 and 30lM, for mouse and human PPARa,
respectively. Differences between their previously reported
LOECs and ours may be influenced by the model used, that is,
variations in the plasmid construct (plasmid sequences, one or
two-plasmid transfection model), cell type utilized, and other
possible differences in the protocol. Regardless, it is noteworthy that our results are within ranges reported in previous
studies by other investigators.
A previous study in our laboratory examined the ability of
PFOA and PFOS to activate PPARa, b/d, and c plasmids in
transfected COS-1 cells (Takacs and Abbott, 2007). That study
demonstrated that PFOA activated the mouse and human
PPARa and PFOS activated the mouse PPARa but not the
human. In the present study, we report lower LOECs for both
PFOA and PFOS in both species and were able to detect
activation of both mouse and human PPARa by PFOS. We
report an LOEC for PFOA of 1 and 10lM for mouse and human, respectively, whereas Takacs and Abbott (2007) reported
LOECs of 10 and 30lM, respectively. In addition, PFOS activated the human PPARa at concentrations of 30lM and
higher, whereas PFOS did not induce activation in the previous
study. This increased sensitivity may be attributable to using
a higher number of replicates of each concentration on each
plate, more plates per assay, and more assays per compound
relative to the previous study.
The concentrations of PFAAs that activated PPARa in this
study are similar to the serum levels of these PFAAs found in
experimental rodents displaying toxicological effects. Gestational exposure of rats and mice to PFOA and PFOS has been
shown to induce full litter resorption, delay eye opening and
preputial separation, inhibit mammary gland and lung development, increase liver weight, and result in postnatal
mortality of pups (Grasty et al., 2003; Lau et al., 2006; White
et al., 2007). Mice exposed to PFOA at doses that were
associated with reduced survival of their offspring (dosed on
gestational day [GD] 117 at 5 mg/kg/day) had serum levels
on postnatal day 22 of 37 ppm for dams and 2225 ppm for
pups (Wolf et al., 2007). After exposure to PFOS from GD1 to
17 at 10 or 20 mg/kg/day, maternal mouse serum levels on
GD18 were 179 and 261 ppm (Lau et al., 2007). These
concentrations are higher than the range of concentrations of
PFOA and PFOS that activated the PPARa in our culture assay
(LOEC: PFOA 0.43 and 4.3 ppm and PFOS 48 and 16 ppm, for
mouse and human, respectively), although the correlation
between these in vivo and in vitro values remains to be
determined. It is also notable that concentrations of PFAAs that
activated the PPARa in this study approach the levels of
PFAAs found in the environment and in human tissues. PFHxS
was measured in house dust at 0.4 ppm (Shoeib et al., 2005),
compared to 4.38 ppm of PFHxS that activated human PPARa
in the current study. PFOA elicited significant PPARa activity
in mouse in our study at 0.43 ppm or 430 ng/ml, ~10 times the
level found in the serum of some human populations (1456
ng/ml, Kannan et al., 2004). Other PFAA (PFBA, PFBS,
PFNA, PFHxA, and PFHxS) that activated the human PPARa
in this study were found in human sera around the world (e.g.,
PFHxS at 28 ppb, Karrman et al., 2007; PFDA at 0.64 ppb and
PFNA at 3.8 ppb, Kuklenyik et al., 2004; reviewed by Lau
et al., 2007). For comparison, in our study, the LOEC for
human plasmid for PFHxS was 4.38 ppm, for PFDA was > 51
ppm, and for PFNA was 2.3 ppm. Some of these compounds
are currently being tested in laboratory animals in vivo for
possible developmental toxicity.
Activation of PPARa may be associated with the developmental or hepatic toxicity mentioned above. PPARa is
found in various organs in the developing fetus (Braissant and
Wahli, 1998). Abbott et al. (2007) demonstrated in knockout
mice that PPARa was necessary for the induction of postnatal
lethality after exposure to PFOA. While it is impossible by this
study to determine whether PPARa activation is involved in
developmental toxicity, the overlap of PPARa activation with
developmental toxicity at similar concentration ranges alerts us
PFAAs ACTIVATE THE MOUSE AND HUMAN PPARa
to the possibility that PPARa may be involved in developmental effects of PFAAs. Activation of PPARa may be a useful
parameter by which to compare PFAA activity and may be an
indicator of PFAA toxicity.
Presented herein is a comprehensive study on the activation of
PPARa in vitro by various perfluorinated compounds. The
transactivational assay developed by Takacs and Abbott (2007)
proved to be a good model for screening PFAAs for PPARa
activation. The highlight of this study is the ability to effectively
compare various PFAAs of different carbon chain lengths and
functional groups across mouse and human species on PPARa
activity. It is important to note that activity in this model only
evaluates the potential for a compound to interact with the PPAR
LBD and activate the reporter and is not necessarily predictive of
the chemical’s ability to produce a toxicological response
in vivo. It is also possible that in an in vivo setting, PFAAs could
produce biological responses that are independent of the PPAR
receptors. However, this culture model was very useful for
making comparisons between PFAAs and between mouse and
human PPAR activity. We have demonstrated that (1) all PFAA
tested (PFBS, PFBA, PFHxS, PFHxA, PFOA, PFOS, PFNA,
and PFDA), except for PFDA with human plasmid, activated the
PPARa in mouse and human plasmids, (2) PFAAs induced
higher activation of PPARa as their carbon backbone chain
length increased, (3) the carboxylates induced higher activation
compared to the sulfonates, and (4) activity of PPARa by
PFAAs was usually higher in the mouse PPARa compared to
the human. This information enables investigators and risk
assessors to identify PFAAs that require more attention or to
select PFAAs for industrial and commercial use that are of less
risk to health and safety.
169
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