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STRUCTURE-ACTIVITY RELATIONSHIPS BETWEEN PERFLUORINATED

CHEMICALS AND MITOCHONDRIAL RESPIRATION RATES

A THESIS
SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL
OF THE UNIVERSITY OF MINNESOTA
BY

Josiah Nathanael Ray

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS


FOR THE DEGREE OF
MASTER OF SCIENCE

Dr. Kendall Wallace

February, 2010

Josiah Nathanael Ray, 2010

Acknowledgements
Id like to thank Dr. Kendall Wallace and James Bjork for their assistance and
advice throughout my graduate career, particularly with my thesis project. You have
spent a great deal of time in teaching me the importance of a careful and methodical
approach to the scientific process, and I greatly appreciate your interest in my
development as a student and as a person. I hope that the work that I present in this thesis
helps to further the research of other students.

I would also like to thank my family for the years of support that they have shown
me while I have been in graduate school. Your love and support has given me the
inspiration that I have needed when I have been discouraged.

Abstract
Perfluorinated chemicals are synthesized compounds that are structurally derived
from hydrocarbons, but have carbon-hydrogen bonds replaced with carbon-fluorine
bonds. They have been shown to inhibit respiration in rat liver mitochondria in vitro. The
objective of this investigation was to explore whether structure-activity relationships exist
for the effects of perfluorinated chemicals on mitochondrial respiration. Freshly isolated
rat liver mitochondria were incubated with increasing concentrations of one of fifteen
different perfluorinated chemicals, after which oxidative phosphorylation was recorded
polarographically with an oxygen electrode. Each analysis was performed with five
concentrations of the respective perfluorinated chemical, and repeated with mitochondria
isolated from five individual animals. The respiration profiles of structurally related
perfluorinated chemicals were then compared to each other. For some perfluorinated
chemicals, increased carbon-chain length corresponded with an increased impact on
mitochondrial respiration. However, it appeared that the effect of chain length was
secondary to the ability of the perfluorinated chemical to efficiently carry protons across
the mitochondrial membrane.

ii

Table of Contents
Section

Page

Acknowledgements.......i

Abstract............ii

Table of Contents................iii

List of Tables .............iv

List of Figures..............v

Introduction..........1

Materials and Methods.......13

Results........21

Discussion......36

Bibliography......47
iii

List of Tables
Table

Table 1

Page

The structures and names of the perfluorinated chemicals investigated in


this study...11

iv

LIST OF FIGURES
Figure

Page

Figure 1

The tracing of a respiration experiment that was performed on


mitochondria that were exposed to PFHXA.............18

Figure 2

The state 3 respiration rates of mitochondria were measured before PFAA


addition (state 3a) and after PFAA addition (state 3b)......23

Figure 3

The comparison of state 4 respiration rates among the PFAAs that were
tested in this study.....28

Figure 4

The comparison of state 3 respiration rates among the PFAAs that were
tested in this study.....29

Figure 5

The comparison of RCR among PFAAs. Each bar indicates the mean +/standard error for five independent mitochondrial preparations...30

.
Figure 6

The impact of PFAAs on ADP:O ratios....31

Figure 7

The comparison of state 4 EC50s among perfluorosulfonamides ....32

Figure 8

The comparison of the EC50s for state 3 respiration rates among the
perfluorosulfonamides ......33

Figure 9

The comparison of RCR EC50s among perfluorosulfonamides ...34


v

Figure 10

The comparison of ADP:O EC50s among perfluorosulfonamides .......35

vi

Introduction
Perfluorinated chemicals are fluorine-substituted hydrocarbons that are
manufactured for use as surfactants, sealants, foams, and other industrial purposes. They
have similar physical characteristics to hydrocarbon-based surfactants: they have limited
volatility and limited solubility in either aqueous or nonpolar solvents. Some of the most
environmentally widespread perfluorinated chemicals are perfluoroalkyl acids (PFAAs),
which tend to be surface-active, have a low pKa, and exist predominantly in the anionic
form. Their manufacture began when 3M scientists produced perfluorooctanoic acid
(PFOA) in 1947 (Prevedouros, Cousins et al. 2006) and perfluorooctane sulfonic acid
(PFOS) in 1952 via the electrochemical fluorination process. Because perfluorinated
chemicals with eight-carbon chain lengths are well-suited for use as surfactants (Lau,
Anitole et al. 2007), PFOA became an important emulsification agent in the production
of Teflon, and PFOS was utilized in the production of firefighting agents in industrial
processes (Lehmler 2005).
The discovery that organic fluorine is present in human blood serum (Taves 1968)
led to the measurement of detectable amounts of PFOA and PFOS in human serum
(Hansen, Clemen et al. 2001). Since that time, PFOA and PFOS have been detected in the
serum of humans and wildlife from around the world (Lau, Anitole et al. 2007; Reagen,
Ellefson et al. 2008). Their serum concentrations were highest in the U.S. and other
developed countries (Kannan, Corsolini et al. 2004), but they were also detected in
uninhabited areas such as the arctic (Martin, Smithwick et al. 2004; Young, Furdui et al.
2007). These compounds are thought to be dispersed globally via transport through the
1

ocean (Prevedouros, Cousins et al. 2006) or through the breakdown of volatile fluorinated
precursors in the atmosphere (Young, Furdui et al. 2007). According to data obtained
from blood donations to the Red Cross in 2005, U. S. citizens had mean human blood
serum concentrations of 15.1 and 2.2 ppb of PFOS and PFOA, respectively (Olsen, Mair
et al. 2007). This is down from 33.1 and 4.5 ppb in 2000 (Olsen, Huang et al. 2005). A
separate study confirmed that PFOS and PFOA blood serum concentrations dropped in
the years 2000 2004 (Calafat, Wong et al. 2007). However, the production of other
perfluorinated chemicals such as telomers and short-chain PFAAs are increasing (Olsen,
Huang et al. 2005; Olsen, Chang et al. 2009). Levels of PFOA and PFOS are far greater
in factory workers that have been occupationally exposed to the compounds, who have
had mean serum levels of 941 and 899 ppb, respectively (Olsen, Logan et al. 2003).
There are two primary reasons for the global presence of PFOA and PFOS in
animals. The strong fluorine-carbon bond confers strong resistance to physical, chemical,
and biological breakdown in these molecules, making them more stable than
conventional hydrocarbon-based surfactants. The carbon-fluorine bond in PFOA and
PFOS is so strong that it is not degraded through any known biological mechanism (Kudo
and Kawashima 2003), causing it to persist in the global environment. Furthermore, the
strength of the carbon-fluorine bond prevents PFOA and PFOS from being metabolized
by animals. PFOA, PFOS, and perfluorohexane sulfonic acid (PFHXS) have geometric
mean serum elimination half-lives of of 3.5 years, 4.8 years, and 7.3 years, respectively
(Olsen, Burris et al. 2007). However, perfluorobutane sulfonic acid (PFBS) is only
retained in humans for about a month (Olsen, Chang et al. 2009).There is evidence that
2

the retention rates of these compounds are influenced by sex and species in rodents
(Kudo, Suzuki et al. 2001; Ohmori, Kudo et al. 2003), but not in humans (Chang, Das et
al. 2008). These differences are due to the estradiol-dependent expression of the organic
ion transporter Oatp1a1 in rodents (Weaver, Ehresman et al.; Andersen, Clewell et al.
2006).
Though occupational health studies found that levels of organic fluorine were
significantly higher in factory workers than the rest of the population , PFOA did not
exist at levels sufficient to induce an association with courses of mortality (Lundin,
Alexander et al. 2009; Sakr, Symons et al. 2009). Dose-response studies that were
performed on rodents indicated that high exposure to PFOA leads to liver, testicular, and
pancreatic cancers in rats (Biegel, Hurtt et al. 2001; Kennedy, Butenhoff et al. 2004).
PFOA has not been shown to induce cancer in occupational health studies (Lundin,
Alexander et al. 2009). However, occupational health studies only provide a limited
assessment of the toxicity of perfluorinated chemicals, because it is possible that factory
workers were not exposed to PFOA or other perfluorinated chemicals at concentrations
necessary to induce a statistical difference in toxicity. In recent years, studies have been
performed on rats, mice, and primates to determine whether perfluorinated chemicals
pose a potential hazard toward humans.
PFOS and PFOA are primarily distributed to the serum, kidney, and liver of
rodents and primates. PFOS and PFOA concentrate in the liver of rodents at low
exposure levels (Kudo, Sakai et al. 2007; Cui, Zhou et al. 2009) , while PFOA tends to
have a higher concentration in the serum of primates (Olsen, Hansen et al. 2003;
3

Butenhoff, Kennedy et al. 2004; Andersen, Clewell et al. 2006), although the
concentration approaches the lower limit of detection..
PFOS and PFOA are able to cross the placenta (Monroy, Morrison et al. 2008),
and these compounds have been shown to affect the development of rat pups in utero
(Luebker, York et al. 2005). Pups that have prenatal exposure to PFOS often die within
one day of birth in a dose-dependent manner (Lau, Butenhoff et al. 2004; Wolf, Fenton et
al. 2007). Mice pups with prenatal exposure to PFOA experience postnatal death as well
(Lau, Butenhoff et al. 2004; Wolf, Fenton et al. 2007). Because one of the characteristics
of prenatal PFOS exposure to rat pups is respiratory distress (Lau, Anitole et al. 2007),
the effect of these compounds on lung maturation was measured. Exposure to PFOS
caused respiration in neonatal rats to become labored (Lau, Thibodeaux et al. 2003).
Further analysis confirmed that exposed rat pups had thickened mucosal membranes and
increased mucus in their lungs (Cui, Zhou et al. 2009), though differentiation of their
lung tissue was normal (Grasty, Bjork et al. 2005). It is possible that PFOS physically
interferes with respiration through its properties as a surfactant (Lehmler, Xie et al.
2006). PFOA induced similar effects in mouse pups (Wolf, Fenton et al. 2007), while
PFBA (perfluorobutanoic acid) did not (Das, Grey et al. 2008).
Prenatal exposure to PFOA and PFOS resulted in important developmental
effects. Rats that survived prenatal exposure to PFOA and PFOS had decreased weight
gain and delayed eye opening rates compared to controls (Case, York et al. 2001). The
serum profiles of the rats indicated a reduction of T4 (thyroxine), but free T4 and TSH
(thyroid stimulating hormone) levels remained the same (Lau, Thibodeaux et al. 2003;
4

Luebker, York et al. 2005). Because exposure to PFOS did not affect TSH levels or
induce symptoms of hypothyroidism (Chang, Thibodeaux et al. 2007; Chang,
Thibodeaux et al. 2008), it is unlikely that the effect of PFOS on prenatal development is
mediated by changes in thyroid hormone.
PFOA also causes changes in the lipid metabolism of rodents. Specifically, it
causes serum triglyceride and cholesterol levels to fall, and causes hepatic triglyceride
and cholesterol levels to rise in rats (Kennedy, Butenhoff et al. 2004). Some occupational
health studies indicated that PFOA may induce minor changes in the intermediary
metabolism of humans (Olsen and Zobel 2007; Sakr, Kreckmann et al. 2007; Costa,
Sartori et al. 2009), though such observations were inconsistent (Olsen and Zobel 2007).
Such changes in serum lipids are consistent with that caused by therapeutic
PPAR agonists, such as the fibrates.
PFOA and PFOS contributed to metabolic dysfunction in rats. Rats that were fed
PFOA developed enlarged livers and reduced body weight (Kennedy, Butenhoff et al.
2004). Similar symptoms were observed in rats and primates that were fed PFOS (Seacat,
Thomford et al. 2002; Seacat, Thomford et al. 2003). The tissue analysis of rats that had
been exposed to PFOS and PFOA revealed cellular damage to the liver and thickened
lung walls (Cui, Zhou et al. 2009). The serum profiles of these animals revealed that they
have decreased serum cholesterol, glucose, and triglyceride levels.
Gene expression profiles of the livers of male rats revealed that genetic transcripts
of many metabolic genes were altered by exposure to PFOA (Guruge, Yeung et al. 2006)
and PFOS (Bjork, Lau et al. 2008). The genes that were affected included those that
5

control lipid, glucose, and cholesterol synthesis (Bjork, Lau et al. 2008; Rosen, Lee et al.
2008; DeWitt, Shnyra et al. 2009). These metabolic changes are induced by an increase
in the nuclear receptor peroxisome proliferater receptor alpha (PPAR) (Bjork, Lau et al.
2008; Rosen, Lee et al. 2008).
PFOA causes testicular, pancreatic, and hepatic tumors in rodents (Biegel, Hurtt
et al. 2001; Kennedy, Butenhoff et al. 2004; Vanden Heuvel, Thompson et al. 2006),
while PFOS has been linked to the incidence of hepatic tumors in rodents (DeWitt,
Shnyra et al. 2009). The tumorigenic effects of PFOA in the liver of rodents have been
linked to PPAR, (Klaunig, Babich et al. 2003; Rosen, Lee et al. 2008). PFOS and PFOA
can activate the human and mouse isoforms of PPAR (Vanden Heuvel, Thompson et al.
2006; Takacs and Abbott 2007), but primary liver cell cultures of rats and humans differ
in their sensitivity toward PPAR activation (Bjork and Wallace 2009). Peroxisome
proliferation has been shown to directly impact mitochondrial bioenergetics (Zhou and
Wallace 1999) and to alter the genetic transcription levels of important metabolic
enzymes (Klaunig, Babich et al. 2003). However, increased levels of cellular vacuoles
and increased liver weights continued to be observed in the PPAR knockout mice
(Wolf, Moore et al. 2008), which indicate that PPAR is not the only causative factor in
the metabolic dysfunction that is associated with PFOA. Other metabolic targets such as
PPAR and CAR (constitutive activated/androstane receptor) may also be implicated in
mitochondrial dysfunction (Rosen, Lee et al. 2008; Walters, Bjork et al. 2009).
Perfluorosulfonates such as PFOS had a less robust effect on PPAR activity than PFOA

(Seacat, Thomford et al. 2003; Vanden Heuvel, Thompson et al. 2006; Takacs and
Abbott 2007; Bjork and Wallace 2009).
PFAAs induce long-chain acyl CoA synthetase in a PPAR-dependent manner
(Ammerschlaeger, Beigel et al. 2004) and a carbon chain-length dependent manner
(Vanden Heuvel, Kuslikis et al. 1991). PFAAs of 6-9 carbons also induce the fatty acid
transporter 1-acyl glycerophosphocholine acyltransferase in hepatocytes .(Kudo, SuzukiNakajima et al. 2006). The induction of long-chain acyl CoA synthetase and 1-acyl
glycerophosphocholine acyltransferase indicate the mechanism by which perfluorinated
chemicals contribute to fatty acid oxidation.
To illustrate how perfluorinated chemicals are thought to induce toxicity through
mitochondrial respiration, the process of oxidative phosphorylation must be described.
Mitochondria have the unique and essential function of converting adenosine diphosphate
(ADP) to adenosine triphosphate (ATP). According the chemiosmotic hypothesis of
mitochondrial respiration (Mitchell 1961), most of the energy that is required for ATP
synthesis comes from the generation of an electrochemical gradient across the inner
mitochondrial membrane. This gradient is generated when the glycolytic product acetyl
CoA reduces the electron carriers FAD and NAD. The terminal complex of the electron
transport chain produces the electrochemical gradient by creating water from oxygen,
which consumes electrons. The flow of protons back across that gradient provides the
energy required for ATP synthase to produce ATP from ADP and inorganic phosphate.
Therefore, the consumption of oxygen is said to be coupled to ATP production.

The insecticide sulfuramid (PFOSA) was found to be associated with the


disruption of mitochondrial respiration in 1990 (Schnellmann and Manning 1990)
through disruption of the inner mitochondrial membrane and neutralization of its pH
gradient. In addition, PFOS was found to increase the cellular membrane fluidity and
disrupt the mitochondrial membrane potential of fish leukocytes at 5- 15 mg/l (Hu, Jones
et al. 2003). Further studies of perfluorinated chemicals indicated that they act on
mitochondrial respiration via three distinct mechanisms (Starkov and Wallace 2002).
Some fluorinated compounds, including PFOS and PFOSAA, affect mitochondria by
directly disrupting the mitochondrial membrane, as might be expected by compounds that
are structurally similar to detergents (Starkov and Wallace 2002; Hu, Jones et al. 2003).
A subset of the perfluorocarboxylates, including PFOA, induce the mitochondrial
permeability transition (MPT), which causes the inner mitochondrial membrane to
become permeable to small molecules (O'Brien and Wallace 2004). The effects of the
MPT include the loss of mitochondrial membrane potential and the efflux of cytochrome
c. Finally, the perfluorosulfonamides can inhibit mitochondria by acting as protonophoric
uncouplers (Schnellmann and Manning 1990; Starkov and Wallace 2002). Such
compounds are immediately recognizable because they cause an immediate increase in
the respiration rates of mitochondria to compensate for the loss of the proton gradient.
Although the toxicity of PFOS and PFOA have been well characterized, the
toxicity profiles of many other perfluorinated chemicals are either unknown or poorly
characterized. The effects of PFBS have been studied in rats and primates, and the
compound is known to be eliminated more rapidly than its longer- chain counterparts
8

(Chengelis, Kirkpatrick et al. 2009; Olsen, Chang et al. 2009). Long-term toxicity studies
indicated that PFBS has minimal impact on the histopathology of rodents, particularly in
comparison to PFOS (Lieder, Chang et al. 2009) PFOSA (perfluorooctanesulfonamide,
or sulfuramid) has been shown to induce reduced cholesterol and lipid production in
rodents in a similar manner as PFOS (Haughom and Spydevold 1992). The
perfluorosulfonamide N-ethyl perfluorooctanesulfonamidoethanol (N-EtFOSE) has also
been investigated in rodents; it is known to induce peroxisome proliferation (Berthiaume
and Wallace 2002) and has been well-characterized as an inhibitor of mitochondrial
respiration (O'Brien and Wallace 2004). To generate a more complete risk assessment of
fluorinated compounds, the NIEHS (National Institute of Environmental Health Sciences)
has funded the current study to determine the relative toxicity that 15 perfluorinated
chemicals have on mitochondrial respiration in vitro. The resulting data will complement
other studies on the structure-activity relationships of perfluorinated chemicals (Starkov
and Wallace 2002; Kudo, Suzuki-Nakajima et al. 2006; Bjork and Wallace 2009).
The toxicity of some perfluorinated chemicals toward mitochondrial respiration
has previously been outlined (Langley 1990; Schnellmann and Manning 1990;
Berthiaume and Wallace 2002; Starkov and Wallace 2002; O'Brien and Wallace 2004).
PFOA, PFOS, N-ETFOSE, and PFOSA are all eight-carbon perfluorinated chemicals that
have been industrially produced for many years. Recently, PFBS, PFHXA, and 8+2
telomers have replaced these molecules because they tend to be eliminated by mammals
more efficiently (Chang, Das et al. 2008; Chengelis, Kirkpatrick et al. 2009; Olsen,
Chang et al. 2009), and because they induce fewer toxicological effects (Lieder, Chang et
9

al. 2009). Although the toxicity of PFBS and PFBA in neonatal rats was significantly
lower than that of PFOS and PFOA, the chemicals still induced enlarged livers in rodents,
albeit at higher doses (Lieder, Chang et al. 2009; Olsen, Chang et al. 2009). It is likely
that the reduced toxicity of these compounds relative to PFOA and PFOS in rodents is
due to a combination of factors, including their rapid elimination rates and reduced
activation of PPAR (Bjork and Wallace 2009). However, the effect of these compounds
on mitochondrial respiration was unknown prior to this study. This study will determine
whether the series of perfluorinated chemicals induce an acute effect on mitochondrial
respiration.
The objective of this study is to determine if the carbon chain length and the
functional groups of perfluorinated chemicals are important determinants of their impact
on mitochondrial respiration rates. The structure-activity relationships that perfluorinated
chemicals have on mitochondrial respiration infer the mechanisms by which they induce
toxicity. For instance, PFDA appears to have a biphasic effect on mitochondrial
respiration: it effects state 4 respiration at low doses and state 3 respiration at higher
doses (Langley 1990). In this study, the relative change in state 3 and state 4 respiration
rates of PFAAs with shorter carbon chain length will be compared to those with longer
carbon chain lengths to determine how PFAAs inhibit mitochondrial respiration. This
study will also compare perfluorosulfonamides of various substituted amino groups.
Although there are well-defined toxicological profiles for PFOS and PFOA, only scant
information is available on other perfluorinated chemicals. This study will provide an
initial characterization of their relative toxicity.
10

Table 1: The structures of the compounds investigated in this study


Perfluoroalkyl carboxylic
acids
Perfluorobutanoic acid

Symbol

Structure

PFBA

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

F
F

O H

Perfluorohexanoic acid

PFHXA

H
O

Perfluorooctanoic acid

PFOA

O H
F
O

Perfluorononanoic acid

PFNA

H
H

Perfluorodecanoic acid

PFDA
F

Perfluorododecanoic acid

PFDOA

Perfluoroalkyl sulfonic
acids
Perfluorobutane sulfonic
acid
Perfluorohexane sulfonic
acid
Perflourooctane sulfonic
acid
Sulfonamides
N-ethyl
perfluorooctanesulfonamido
ethanol

PFBS

F
F
F

PFHXS

F
F

F
F

F
F

O
S

OH

O
F

F
F
F

PFOS

F
F

F
F

F
F

F
F

F
F

O
S

OH

O
F

NEtFOSE

O
S

OH

O
O

O
S

F
F

N
CH3

11

N-methyl
perfluorooctanesulfonamido
ethanol

NMeFOSE

O
S

CH3
N

O
O

N-ethyl
perfluorooctanesulfonamido
acetate

PFOSAA
F

H3C
O
S

O
OH

N-methyl
perfluorooctanesulfonamido
acetate

M570

O
S

CH3
N

O
O

Perfluorooctanesulfonamido
acetate

M556

O
S

NH

O
O

Perfluorooctanesulfonamide

PFOSA

S
F

Positive controls
Rotenone

Rotenone

CH3

NH2

CH3

O
O

O
O

CH2
H3C

Carbonyl cyanide-ptrifluoromethoxy
phenylhydrazone

FCCP

T-butyl hydroperoxide

TBHP

O
N

NH

CH3
H3C

O
CH3

OH

12

Materials and Methods


Isolation of Hepatocytes
Male Sprague-Dawley rats that weighed between 150-200 grams were obtained
from Charles River Laboratories in Portage, MI. They were maintained in a climatecontrolled facility at room temperature, with exposure to 12 hours of light each day. The
rats were fed Purina lab chow #5001 and were provided with unlimited access to food
and water. The rats were acclimated within the facility for time periods that ranged from
three days to two weeks. When mitochondria were required for experimentation, a rat
weighing between 175 and 300 grams was fasted for 12-24 hours. The next day, it was
killed by being asphyxiated with CO2, then immediately decapitated to ensure that liver
metabolism was not affected. The liver was excised and placed in an ice-cold isolation
buffer that consisted of 200 mM mannitol, 10 mM sucrose, 5 mM HEPES buffer, and 1
mM EGTA, pH 7.4. Approximately 2 grams of the excised liver was minced and washed
repeatedly with this buffer to remove as much blood from the tissue as possible.
Homogenization of the minced liver was carried out with a Potter-Elvhjam
Teflon/glass homogenizer. The tissue was diluted in a 1:8 ratio in the isolation buffer,
then homogenized at low speed, with 3-5 passes. The resulting slurry was centrifuged at
1000 g for 10 minutes at 4 C to remove cellular debris. The supernatant was recovered
and centrifuged at 10,000 g for 10 minutes at 4 C to isolate the mitochondrial fraction,
which existed as a pellet at the base of the centrifuge tube. Free fatty acids were removed
from the mitochondria by suspending the mitochondrial fraction and centrifuging it at
10,000 g again in the aforementioned isolation buffer that was supplemented with 1
13

mg/ml fatty acid-free BSA. The supernatant was removed, and the mitochondrial pellet
was resuspended in a washing buffer with a pH of 7.4 that consisted of 200 mM
mannitol, 10 mM sucrose, and 5 mM Hepes buffer to remove the fatty-acid free BSA.
The suspension was centrifuged at 10,000 g. The mitochondria were resuspended in fresh
washing buffer and centrifuged again at 10,000 g. The supernatant was removed, and the
mitochondrial pellet was resuspended in 0.5 ml of washing buffer before being
transferred to a 1.5 ml Eppendorf tube. The mitochondria were kept on ice for the
duration of the experiment. Under these conditions, mitochondria yielded consistent
respiration rates for roughly six hours.

Respiration protocol
The respiration rates of the mitochondria were measured with a Clark-type
oxygen electrode that was inserted in a 1.5 ml water-jacketed respiration chamber. The
temperature of the respiration chamber was maintained at 30 C with a thermostatcontrolled Haake D3/L water bath. The dissolved oxygen content of the respiration
chamber was subsequently recorded with a chart recorder that was set to run at 10
millimeters per minute. The electrode was equilibrated in 1.5 mls respiration buffer (130
mM sucrose, 59 mM KCl, 2.5 mM KH2PO4, 5 mM Hepes buffer, pH 7.4) for 30 minutes.
The isolated mitochondrial protein was quantified by the modified Bradford assay
with BSA (bovine serum albumen) as a standard. After the electrode had been
equilibrated, 0.75 mg of the mitochondrial protein was added to the respiration chamber
to yield a final concentration of 0.5 mg/ml. A solution of 1 mM glutamate and 1 mM
14

malate in 15 l respiration buffer was added to the chamber. The chamber was then
sealed and the respiration rates were recorded (Zor 1996).

Respiration assays
Respiration states: To determine the mechanism by which the perfluorinated
chemicals under investigation interfere with mitochondrial respiration, their impact on
oxygen consumption was measured. A chart of a representative respiration experiment is
shown below (figure 1). State 3 and state 4 respiration rates refer to the respiration rates
in the presence and absence of ADP, respectively. The initial respiration rate (state 4a)
began as soon as the respiration chamber was sealed. State 3a respiration was initiated
when ADP (375 moles) was added to the respiration chamber with a syringe for a final
concentration of 250 M (state 3a). The state 4 respiration rates (state 4b) resumed when
ADP was exhausted. The ratio of state 3a: 4b respiration rates yielded the primary
respiratory control ratio (RCR 1) of the untreated mitochondria. If the mitochondria
remained intact after the isolation procedure, the primary RCR ranged from 4 to 7. Only
isolated mitochondria that generated primary RCRs over 4 were used in this study. If the
mitochondria yielded a primary RCR under 4, the experimental tests of these
mitochondria were terminated.
After ADP had been depleted and the initial respiration rates were established
(state 4b), 15 l of one of the concentrations of a perfluorinated chemical (figure 1) was
added to the solution via a syringe that was specifically used for the addition of
perfluorinated chemicals. This initiated the state 4c respiration rate. A final concentration
15

of 250 M ADP was added again to the solution (state 3b) and was consumed by the
mitochondria until it was exhausted (state 4d). The ratio of state 3b: 4d respiration rates
yielded the secondary RCR ratio (RCR 2). A comparison of RCR 2 to RCR 1 was used to
determine the effect that perfluorinated chemical concentration has on mitochondrial
respiration.
After state 4d was established, the respiration chamber was emptied and washed
repeatedly with ethanol and water. The syringes that were used to deliver ADP and the
perfluorinated chemicals were also cleaned with ethanol and water. Any possible
carryover contamination was normalized by the measurement of respiration rates before
and after perfluorinated chemical addition (state 4d: state 4b). The above process was
repeated with a different concentration of the same perfluorinated chemical to determine
the respiration rates for a total of five concentrations of each individual perfluorinated
chemical. The zero concentration included only 15 l of the solvent (DMSO). All of the
respiration rates that were recorded from a mitochondrial extract were calculated and
subsequently stored in a Microsoft Excel spreadsheet.
The five standard concentrations of each perfluorinated chemical that were
measured in the respiration experiments were predetermined by a range-finding assay
that had previously been conducted. The range-finding assays were performed in the
same manner as the respiration experiments, though only the RCRs were recorded and
compared. The goal of the range-finding experiments was to determine the concentration
of perfluorinated chemical that would decrease the RCR by roughly 50%. Two higher

16

and two lower standard concentrations were then selected, so that the highest standard
concentration was roughly one order of magnitude higher than the lowest.
In summary, the respiration data that was collected for each perfluorinated chemical was
generated from mitochondria that were isolated from five individual rat livers, which
represented five replicates of each dose-response assay. Each replicate had its own zero
concentration control. Each mitochondrial extract, in turn, was exposed to five standard
perfluorinated chemical concentrations. Fifteen perfluorinated chemicals and three
positive controls were investigated in this study, which were in addition to 15 l of the
vehicle control (DMSO) that was included in each experiment.

17

Respiration chart PFHXA


90%

250 M
ADP

oxygen

80%
250 M
ADP

3a
4b

4c

70%

250 M
PFHXA

60%

3b
4d

50%
2

10

12

14

minutes

Figure 1: The tracing of a respiration experiment that was performed on mitochondria


that were exposed to PFHXA. It illustrates the various respiration states of mitochondria
and how they are affected by PFAAs. In the above experiment, 250 M PFHXA was
added to the respiration chamber to generate the rates for state 3b and 4d respiration after
the rates of state 3a and state 4b respiration were established. All respiration rates are
expressed in units of nmol O2/min/mg protein.

18

ADP:O ratios: To determine whether the perfluorinated chemicals inhibit


oxidative phosphorylation, ADP:O ratios were calculated (Estabrook 1967). The state 4b
and state 4d respiration rates had to be extrapolated to determine what the respiration
rates of mitochondria would have been if ADP had not been added to the mitochondria.
The amount of oxygen that was consumed in the process of phosphorylating the added
ADP was calculated before and after the addition of the perfluorinated chemical in each
respiration experiment and were expressed as the percent decrease in the oxygen content
of the respiration chamber that was induced by ADP. That ratio was multiplied by the
standard oxygen content of respiration buffer (1380 nanoatoms oxygen per ml) and
volume of the respiration chamber (1.5 ml) to yield the microatoms of oxygen consumed
by ADP. Finally, this value was divided by the amount of ADP that had been added to
the solution (375 M) to yield an ADP:O ratio (M ADP/atoms oxygen utilized). Like
the other respiration data, ADP:O ratios were calculated before and after the addition of
each perfluorinated chemical to generate a dose-response curve for each class of
perfluorinated chemicals.
The perfluorinated chemicals that were included in the present study varied
greatly in the effective concentrations at which they affect mitochondrial respiration. The
standard concentrations of the perfluorinated chemicals that were administered to
mitochondria in this study were based on those used by Starkov and Wallace (Starkov
and Wallace 2002) and by subsequent rangefinding studies that were performed as
described above. Due to the hydrophobic nature of perfluorinated chemicals, they have
limited solubility in water. Therefore, all the standard concentrations of the PFCs were
19

initially dissolved in DMSO, then diluted 1:100 in the respiration buffer during the
experimental assay.

Statistical Analysis
The comparison of the perfluorinated chemicals to one another was done via
determination of their dose-dependent slopes, which were expressed in charts with the
mean +/- standard error. To determine if the perfluorinated chemicals had a significant
effect on state 3 respiration rates, state 4 respiration rates, RCR, and ADP:O ratios, the
data was analyzed with a paired t-test for independent means. To determine whether the
respiration rates of perfluorinated chemicals differed from each other, the regression
analysis and EC50 of each perfluorinated chemical was compared via Dunnetts post hoc
test for a completely randomized ANOVA with independent means (Dunnett 1955).

20

Results
Respiration Charts: In an effort to determine the dose ranges for which rat liver
mitochondria are sensitive to perfluorinated chemicals, respiration profiles were
generated for each of these compounds as well as positive control compounds. As
mentioned previously, the dose response profile for each perfluorinated chemical was
based on the five mitochondrial preparations that were isolated from individual rats. Each
respiration profile consisted of five respiration plots, one for each of five concentrations
of the perfluorinated chemical, as well as a zero concentration that contained only the
solvent DMSO (figure 1).
The analysis of respiration data began with the determination of initial state 3 and
state 4 respiration rates (before exposure to perfluorinated chemicals). Initial respiration
rates fluctuated somewhat throughout the respiration assay (figure 2a), even though they
were obtained from the same mitochondrial preparation. This was because the isolated
mitochondria tended to adhere to the surface of the micropipette during their transfer to
the respiration chamber, which led to slight variability in the amount of the mitochondrial
protein that was actually added to the respiration chamber. The mitochondrial quality also
tended to decrease with time, which had the same effect as reducing the amount of
available mitochondrial protein. Because increased mitochondrial protein levels
proportionally increase basal respiration rates, any differences in mitochondrial protein
were reflected in the variability in the plots for state 3a and state 4b respiration rates.
Finally, the amount of dissolved oxygen in the respiration chamber decreased throughout
each run, which tended to decrease respiration rates.
21

Due to the variability in initial state 3 and state 4 respiration rates, the standard
error of mitochondria that were exposed to perfluorinated chemicals was increased, and
caused the dose response trends of exposed mitochondria (Figure 2b) to become less
linear. To eliminate some variability in state 3b and 4d respiration rates, these rates were
paired with the initial respiration rates that were generated immediately before the
addition of the perfluorinated chemicals. State 3b/3a and 4d/4b ratios were generated and
plotted against increasing perfluorinated chemical concentration (Figure 2c). This
technique was also used to determine the impact of perfluorinated chemicals on state 4
respiration, ADP:O ratios, and RCR. These ratios were determined for each of the five
mitochondrial extracts that were generated per perfluorinated chemical.

22

Chart A
y = -2.2571x + 149.73
R2 = 0.101

-1

nmol O2 min mg protein

-1

PFHXA 3a
200
150
100
50
0
1

Replicate

Chart B
y = -0.0399x + 111.79

-1

nmol O2 min mg protein

-1

PFHXA 3b

R2 = 0.6152

140
120
100
80
60
40
20
0
0

200

400

600

800

1000

1200

uM PFHXA

Chart C
y = -0.0002x + 0.7683
R2 = 0.8195

state 3 treated/control

PFHXA 3b/3a
1.00
0.80
0.60
0.40
0.20
0.00
0

200

400

600

800

1000

1200

uM PFHXA

Figure 2: The state 3 respiration rates of mitochondria were measured before


perfluorinated chemical addition (state 3a) and after perfluorinated chemical addition
(state 3b). To normalize the data, ratios of state 3b/3a respiration rates were generated at
each dose response point. This paired data had the effect of normalizing dose response
trends (Chart C). The linear regression slope (chart C) was determined for each of five
independent mitochondrial preparations that were exposed to the same perfluorinated
chemical (data not shown).
23

Dose response trends:


The relative impact of perfluorinated chemicals on mitochondrial respiration
could not be directly compared because each perfluorinated chemical was administered to
mitochondria at a different concentration range. To address this problem, the slopes of
the linear regression lines that were generated for 3b/3a, 4d/4b, 4d/4c, ADP:O, and RCR
ratios were plotted as a function of perfluorinated chemical concentrations. These slopes,
which are expressed in units of change in respiration per M of perfluorinated chemical,
allowed the perfluorinated chemicals to be directly compared to one another. Because
five mitochondrial extracts were tested for each perfluorinated chemical, the regression
slopes for each extract were averaged +/- standard deviation (figure 3a).
The dose response rates only take into account the slope of the regression line for
the dose response assays. They do not include the y-intercept data from those assays. As
a result, it is possible for two perfluorinated chemicals to have the same dose response
rates, but different EC50s.
To address this concern, the impact of perfluorinated chemicals on mitochondrial
respiration was also determined by generating the EC50, which is the perfluorinated
chemical concentration that alters mitochondrial respiration by 50%. The EC50 was
calculated by solving for the equation of the linear regression lines for a y= 0.5. The
EC50s of the five respiration experiments that were performed per perfluorinated
chemical were averaged +/- the standard deviation for each perfluorinated chemical
(figure 3b). Unfortunately, relatively flat regression slopes can skew the data by yielding

24

an exceptionally high EC50. To prevent this effect, EC50s that were 2x greater than any
others were omitted from the calculation of the EC50 of a particular compound.

Respiration data analysis

Positive Controls

St4

St3

RCR

ADP:O

Uncoupler (FCCP)

increase

decrease

decrease

decrease

Inhibitor (Rotenone)

decrease

decrease

decrease

no change

FCCP (carbonyl cyanide- p-trifluoromethoxyphenylhydrazone) is a classic


uncoupler of oxidative phosphorylation. It increases mitochondrial state 4 respiration
rates, but decreases state 3, RCR, and ADP:O ratios. Rotenone is a classic inhibitor of
oxidative phosphorylation. It decreases mitochondrial state 3 respiration rates, state 4
respiration rates, and RCR, but has no effect on ADP:O ratios.
Perfluorinated chemicals: All of the PFAAs except for PFBS and PFHXS
significantly altered state 3 respiration rates (figure 3). Exposure to PFDA, PFDOA, and
PFOS induced a greater change in state 3 respiration rates than PFAAs with six or fewer
carbons. The EC50s of PFAAs with more than six carbons were significantly smaller
than those with fewer than six carbons. PFOS also had a significantly stronger impact on
state 3 respiration rates than any of the other carboxylic or sulfonic PFAAs that were
tested.

25

The sulfonamidoacetates were more potent inhibitors of state 3 respiration rates


than the other perfluorinated chemicals that were tested in this study, as most of them had
EC50s under 50 M (figure 8). PFOSA was excluded from the EC50 analysis because it
did not have a significant effect on state 3 respiration rates. The EC50 data had less
variability than the regression data due to the exclusion of outliers.
All of the above PFAAs except PFBA, PFHXA, and PFBS significantly increased
state 4 respiration rates. The PFAAs with carbon chain lengths greater than 6 had a potent
effect on state 4 respiration, but were not significantly different from each other. Among
those compounds, the effect of carbon chain length on state 4 respiration rates was nearly
as robust as its effect on state 3 respiration rates. PFDOA and PFDA induced
significantly greater state 4 respiration rates in mitochondria than PFBA and PFHXA.
The perfluorosulfonamides had a robust effect on state 4 respiration in
mitochondria. PFOSA increased state 4 respiration significantly more than any other
perfluorinated chemical that was tested in this study, with an EC50 of 2 M. PFOSAA
also had a robust effect on state 4 respiration, but the other perfluorosulfonamides had
EC50 that were similar to those that were observed during state 3 respiration.
Each of the perfluorinated chemicals that were tested in this study had a
significant effect on the RCR of mitochondria. The RCR data had less standard error than
the other respiration data because the data was normalized to account for the decrease in
respiration rates that was caused by decreasing oxygen availability. PFAAs with over six
carbons decreased the RCR of mitochondria more than PFAAs with fewer than six

26

carbons. PFOS had a significantly stronger effect on RCR than the perfluorinated
carboxylic acids.
Of the perfluorinated chemicals included in this study, the perfluorosulfonamides
PFOSA, PFOSAA, and M570 had the greatest effect on RCR. These compounds caused a
significantly greater decrease in RCR than M556, N-EtFOSE. N-MeFOSE was the
weakest inhibitor of the perfluorosulfonamides that were investigated in this study.
On average, ADP:O ratios of perfluorinated chemicals with over six carbons were
positively impacted by increased perflouroalkyl acid concentration. ADP:O ratios tended
to be highest at the maximal exposure to perfluorinated chemicals. This increase
indicated that when mitochondria were exposed to perfluorinated chemicals, more
oxygen had to be consumed to utilize all of the exogenous ADP. After the highest
standard concentrations of PFDA and PFDOA were exposed to mitochondria, the ADP:O
ratios became significantly higher than controls. Because this indicated that exogenous
ADP was not completely consumed by the mitochondria, the respiration data for the
highest concentrations of PFDA and PFDOA were eliminated.
When exposure to perfluorinated chemicals resulted in ADP:O ratios over three,
the resulting respiration data were discarded from the data analysis. None of the
perfluorosulfonamides caused the ADP:O ratios of mitochondria to increase significantly.
The standard error of the ADP:O ratio calculations was quite high, because their doseresponse trends tended to be nonlinear. Although uncouplers tend to cause the ADP:O
ratios of mitochondria to fall, none of the perfluorosulfonamides significantly reduced the
coupling efficiency of mitochondria.
27

A.
4d/4b dose rates

% change/M PFAA

0.008
*

0.006

*
0.004
0.002
0
-0.002
PFBA

PFHXA

PFOA

PFNA

PFDA

PFDOA

PFBS

PFHXS

PFOS

B.
4d/4b dose rates

% change/M PFAA

0.35
0.3

0.25
0.2
0.15
0.1
0.05
0
PFOSA

PFOSAA

m570

m556

N-EtFOSE

N-MeFOSE

Figure 3: The comparison of state 4 respiration rates among the perfluorinated chemicals
that were tested in this study. Each bar indicates the mean +/- standard error for five
independent mitochondrial preparations. Asterisks indicate perfluorinated chemicals with
a significantly different dose-dependent effect on state 4 respiration rates than PFOA or
PFOS (figure 3a) or M570 (figure 3b).

28

A.
3b/3a dose rates
% change/M PFAA

0.002
0
*

PFBS

PFHXS

-0.002
-0.004
-0.006
-0.008
PFBA

PFHXA

PFOA

PFNA

PFDA

PFDOA

PFOS

B.

3b/3a dose rates


PFOSA

PFOSAA

M570

M556

N-EtFOSE

N-MeFOSE

% change/M PFAA

0.02
0.01
0
-0.01

*
-0.02
-0.03
-0.04
-0.05

Figure 4: The comparison of state 3 respiration rates among the perfluorinated chemicals
that were tested in this study. Each bar indicates the mean +/- standard error for five
independent mitochondrial preparations. Asterisks indicate perfluorinated chemicals with
a significantly different dose-dependent effect on state 3 respiration rates than PFOA or
PFOS (figure 4a) or m570 (figure 4b).

29

A.
RCR 2/ RCR 1 dose rates

% change/M PFAA

0
-0.002

PFBS

PFHXS

-0.004
*

-0.006
-0.008
-0.01
PFBA

PFHXA

PFOA

PFNA

PFDA

PFDOA

PFOS

B.

RCR 2/RCR 1 dose rates

% change/M PFAA

PFOSA

-0.02

PFOSAA

M570

M556
*

N-EtFOSE
*

N-MeFOSE
*

-0.04
-0.06
-0.08
-0.1
*
-0.12

Figure 5: The comparison of RCR among perfluorinated chemicals. Each bar indicates
the mean +/- standard error for five independent mitochondrial preparations. Asterisks
indicate perfluorinated chemicals with a significantly different dose-dependent effect on
RCR than PFOA or PFOS (figure 5a) or m570 (figure 5b).

30

A.
ADP:O ratio dose rates

% change/M PFAA

0.025
0.02
0.015
0.01
0.005
0
-0.005
PFBA

PFHXA

PFOA

PFNA

PFDA

PFDOA

PFBS

PFHXS

PFOS

B.

% change/M PFAA

ADP:O dose rates


0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0

PFOSA

PFOSAA

m570

m556

N-EtFOSE

N-MeFOSE

Figure 6: The impact of perfluorinated chemicals on ADP:O ratios. Each bar indicates the
mean +/- standard error for five independent mitochondrial preparations. Asterisks
indicate perfluorinated chemicals with a significantly different dose-dependent effect on
ADP:O ratios than PFOA or PFOS (figure 5a) or m570 (figure 5b).

31

A.

4d/4b EC50
2500

M PFAA

2000

1500

1000
500
0
PFBA

PFHXA

PFOA

PFNA

PFDA

PFDOA

PFBS

PFHXS

PFOS

B.
4d/4b EC50
250

M PFAA

200

150
100
50

0
PFOSA

PFOSAA

M570

M556

N-EtFOSE

N-MeFOSE

Figure 7: The comparison of state 4 EC50s among perfluorinated chemicals. Each bar
indicates mean +/- standard error for five independent mitochondrial preparations.
Asterisks indicate perfluorinated chemicals with a significantly different dose-dependent
effect on state 4 respiration rates than PFOA or PFOS (figure 7a) or m570 (figure 7b).

32

A.

3b/3a EC50
2000

M PFAA

1500

*
*

1000
500
0
PFBA

PFHXA

PFOA

PFNA

PFDA

PFDOA

PFBS

PFHXS

PFOS

B.
3b/3a EC50
300
*

M PFAA

250
200
150

100
*

50
0
PFOSA

PFOSAA

M570

M556

N-EtFOSE

N-MeFOSE

Figure 8: The comparison of the EC50s for state 3 respiration rates among the
perfluorinated chemicals that were analyzed in this study. Each bar indicates mean +/standard error for five independent mitochondrial preparations. Asterisks indicate
perfluorinated chemicals with a significantly different dose-dependent effect on state 3
respiration rates than PFOA or PFOS (figure 8a) or m570 (figure 8b).

33

A.
RCR 2/RCR 1 EC50
1200
1000
M PFAA

800

600
400
200

0
PFBA

PFHXA

PFOA

PFNA

PFDA

PFDOA

PFBS

PFHXS

PFOS

B.

M PFAA

RCR 2/RCR 1 EC50


160
140
120
100
80
60
40
20
0

*
*
*
PFOSA

PFOSAA

M570

M556

N-EtFOSE

N-MeFOSE

Figure 9: The comparison of RCR EC50s among perfluoroalkyl acids. Each bar indicates
the mean +/- standard error for five independent mitochondrial preparations. Asterisks
indicate a significantly different dose-dependent effect on RCR than PFOA or PFOS
(figure 9a) or m570 (figure 9b).

34

A.
ADP:O EC50
3000

M PFAA

2500
2000
*

1500
1000

500
0
PFBA

PFHXA

PFOA

PFNA

PFDA

PFDOA

PFBS

PFHXS

PFOS

B.
ADP:O EC50
500

M PFAA

400

300
200
*

100
0

*
PFOSA

*
PFOSAA

M570

M556

N-EtFOSE

N-MeFOSE

Figure 10: The comparison of ADP:O EC50s among perfluoroalkyl acids. Each bar
indicates the mean +/- standard error for five independent mitochondrial preparations.
Asterisks indicate a significantly different dose-dependent effect on ADP:O ratios than
PFOA or PFOS (figure 10a) or m570 (figure 10b).

35

Discussion
Exposure to high doses of PFOS and PFOA have been associated with acute
toxicity in rodents (Kudo and Kawashima 2003; Kudo, Suzuki-Nakajima et al. 2006;
Lau, Anitole et al. 2007), and the liver has been established as the primary target of their
toxicity (Kudo, Suzuki-Nakajima et al. 2006; Cui, Zhou et al. 2009). In vivo studies have
shown that exposure of rodents to PFOA and PFOS alters the transcription of several
metabolic genes (Vanden Heuvel, Thompson et al. 2006; Bjork, Lau et al. 2008; Walters,
Bjork et al. 2009). Although both PFOA and PFOS interfere with mitochondrial oxidative
phosphorylation in isolated rat liver mitochonria,, there is limited information on the
mitochondrial toxicity of other perfluorinated chemicals. In an effort to determine the
relative in vitro mitochondrial toxicity of these compounds, this study measured the
direct effect that fifteen perfluorinated chemicals have on mitochondrial respiration in the
liver. A second objective was to explore the structural determinants of the potency and
mode of action of perfluorinated chemicals on mitochondrial respiration.
The most potent perfluorinated chemicals such as PFOSA, stimulate
mitochondrial respiration by uncoupling the proton gradient in a manner similar to the
potent mitochondrial uncoupler FCCP (Starkov and Wallace 2002). Other perfluorinated
chemicals such as PFOA, have a less pronounced effect on mitochondrial respiration but
have a broader environmental distribution (Quinones and Snyder 2009). Because the
toxicological effect of these compounds on humans is not fully understood, the
determination of their physiological impact is important for the assessment of their
safety.
36

The comparison of perfluorinated chemicals with one another included the


analysis of the correlation of structure-activity relationships on mitochondrial respiration,
specifically the impact of carbon chain length among the PFAAs and the electronwithdrawing group of perfluorosulfonamides. An understanding of the structure-activity
relationship between perfluorinated chemicals will provide a basis for determining the
risk that they present and will shed light on whether the manufacture of alternative
compounds such as short-chain PFAAs lessen the potential impact on mitochondrial
respiration.
The positive controls TBHP, rotenone, and FCCP were tested to demonstrate that
the methods used can distinguish the different mechanisms by which perfluorinated
chemicals interfere with mitochondrial respiration. These compounds helped to determine
whether the respiration profiles that were generated in the present study agreed with
currently accepted mode of action of known compounds and helped to determine the
mechanism by which perfluorinated chemicals interfere with mitochondrial respiration
through comparison of their respiration profiles. Rotenone inhibits complex II of the
electron transport chain, which in turn inhibits both state 3 and state 4 NADH dependentrespiration. TBHP induces the mitochondrial permeability transition, and FCCP
uncouples mitochondrial respiration from ATP synthesis, which serves to increase both
state 3 and state 4 respiration rates. The respiration profiles of these inhibitors were all
consistent with their known mechanisms of action on mitochondrial respiration.

37

Perfluorinated sulfonic and carboxylic acids: The analysis of regression slopes


indicated that carboxylic and sulfonic acids with progressively longer carbon chain
lengths cause an increasingly greater inhibition of the state 3 respiration of mitochondria
(figure 3). The EC50 analysis also indicated that PFAAs with carbon chain lengths over
six have increasing potency on state 3 respiration rates, and that such compounds exert a
significantly stronger effect than their short-chain counterparts. However, the structureactivity relationship of these compounds was not linear; there was a clear dichotomy
between the potency of PFAAs with carbon chains under six and those with longer
carbon chain lengths. The EC50 analysis did not distinguish between the relative potency
of PFAAs with carbon chain lengths of six or less.
The respiration data did not indicate why PFAAs with carbon chain lengths over
six have a significantly greater effect on state 3 respiration than PFAAs with shorter
carbon chain lengths. Among PFAAs with the same functional group, it is likely that
PFAAs with carbon chains of six or less do not readily cross the inner mitochondrial
membrane. Endogenous fatty acids are esterified by long-chain acyl CoA synthetase and
subsequently transferred into the mitochondria via carnitine acyltransferase (Vanden
Heuvel, Kuslikis et al. 1991). Although PFAAs are structurally similar to fatty acids, they
are not substrates for acyl CoA synthetase (Kuslikis, Vanden Heuvel et al. 1992) or for
carnitine acyltransferase (Vanden Heuvel, Kuslikis et al. 1991). It has also been
hypothesized that perfluorinated chemicals may be transported across the inner
mitochondrial membrane via passive diffusion in a carbon-chain length dependent
manner (Xie, Bothun et al.; Lehmler, Xie et al. 2006). Regardless of the mode of action
38

by which carboxylic and sulfonic PFAAs inhibit state 3 respiration rates, it appears that
they must have over six carbons to have an important effect on state 3 respiration.
Interference with state 4 respiration, like state 3 respiration, was affected by
PFAA carbon chain lengths. The regression analysis and EC50 data indicated that
perfluorocarboxylic acids with carbon chain lengths over six increased state 4 respiration
significantly more than their shorter chain counterparts. Because increased state 4
respiration is indicative of disruption of the proton gradient across the inner
mitochondrial membrane, perfluorocarboxylic acids with carbon chain lengths over six
may act as mild uncouplers, either directly or via induction of the MPT.
The sulfonic acids also moderately increased state 4 respiration rates. Like the
carboxylic acids PFHXA and PFBA, the sulfonic acids PFHXS and PFBS exerted a mild
effect on state 4 respiration, with EC50 concentrations of roughly 1 mM (c.a., 15-20
ppm) which is several orders of magnitude greater than the reported environmental
human serum concentrations of roughly 1 ppb (Calafat, Wong et al. 2007). PFOS had a
similar effect on state 4 respiration rates as PFOA at most concentrations, though the
results were more variable (figure 3). However, PFOS inhibited state 4 respiration to a
significantly greater degree than PFOA at its highest concentration (data not shown).
Mitochondria are particularly sensitive to the ionic strength of detergents (Starkov and
Wallace 2002); therefore, PFOS may be a stronger inhibitor of state 3 respiration due to
its higher ionic strength. In vitro experiments have indicated that PFOS has the ability to
degrade the outer mitochondrial membrane (Hu, Jones et al. 2003).

39

The ADP:O ratios indicate whether a PFAA caused the mitochondria to consume
more oxygen to phosphorylate a given amount of ADP. A decrease in ADP:O is
indicative of an uncoupler of oxidative phosphorylation because more oxygen must be
consumed to maintain the electron gradient across the inner mitochondrial membrane
(Estabrook 1967). The analysis of ADP:O ratios in this study was complicated by the
large amount of variability in the effect of PFAAs on respiration. This was partially due
to the variation in the standard concentrations of PFAAs that were exposed to
mitochondria, but was primarily caused by a dichotomy in the mechanism of inhibition
by many PFAAs. The longer-chain PFAAs (PFOA, PFNA, PFDA, PFDOA, and PFOS)
tended to sharply decrease state 3 respiration at high concentrations. These trends tended
to be accompanied by higher ADP:O ratios, which may have been an artifact of the
method of defining the transition between state 3 and state 4 respiration..
The ADP:O ratios of mitochondria were significantly increased by the highest
standard concentrations of PFDA (200 M) and PFDOA (100 M). Because ADP:O
ratios of 3 are considered to be the highest level that is biologically achievable (Estabrook
1967), the most likely cause of such artificially high ADP:O ratios is that the
mitochondria were unable to phosphorylate all of the ADP in the respiration chamber
before the reaction was terminated. Therefore, the respiration data that was gathered at
these concentrations were omitted from the data analysis.
The EC50s for many of the PFAAs that were investigated in this study are
consistent with those reported by other studies (Starkov and Wallace 2002). All of the
PFAAs that were included in this study showed a significant impact on RCRs. However,
40

the range in the EC50 RCRs of the PFAAs varied greatly, from 800 M for PFAAs with
carbon chain lengths of six or fewer carbons to around 50 M for the PFAAs with longer
carbon chain lengths. The mechanism behind this divergence was not explored in the
current study, but current knowledge of mitochondrial bioenergetics leaves a possible
explanation. PFAAs with longer carbon chain lengths more closely resemble endogenous
fatty acids and may be more likely to transverse or associate with the mitochondrial
membrane because they are more hydrophobic. Smaller PFAAs, on the other hand, tend
to have greater water solubility, and therefore may not be as likely to cross the
mitochondrial membrane.

Perflurosulfonamides
This study indicated that perfluorosulfonamides exert a significant effect on the
state 3 respiration of rat liver mitochondria (figure 8). This effect may be caused in part
by the mitochondrial permeability transition (MPT), which is characterized by the
mitochondrial swelling. The MPT causes the rapid efflux of mitochondrial contents,
including cytochrome c (Panaretakis, Shabalina et al. 2001). Although the MPT has been
shown to be induced by the N-acetyl perfluorosulfonamides PFOSAA and m556, it is not
induced by PFOSA or N-EtFOSE (Starkov and Wallace 2002), which indicates that the
N-acetyl group is an important determinant of MPT-mediated mitochondrial dysfunction.
(O'Brien and Wallace 2004). Because the N-acetyl perfluorosulfonamides PFOSAA,
m570, and m556 had a significantly greater impact on state 3 respiration rates than the
perfluorosulfonamides PFOSA, N-EtFOSE, or N-MeFOSE, this study confirms that the
41

MPT may be an important mechanism of their impact on state 3 respiration rates. Since
all of the perfluorosulfonamides have some impact on state 3 respiration, it is likely that
perfluorosulfonamides also inhibit state 3 respiration in a nonspecific manner that is
similar to that observed in PFOA and PFOS, which do not inhibit the MPT (O'Brien and
Wallace 2004).
The perfluorosulfonamides strongly increased state 4 respiration rates in a dosedependent manner. As expected, their impact on 4 respiration rates was directly
proportional to the strength of their electron withdrawing groups. The powerful increase
in state 4 respiration rates that was caused by perfluorosulfonamides indicates that they
act as potent uncouplers of the mitochondrial membrane. For reference, the calculated
EC50 for PFOSA was approximately 5 M or 100 ppb.
The toxicological significance of the perfluorosulfonamides as uncouplers of
mitochondrial respiration rather than inhibitors of mitochondrial respiration is somewhat
unclear. The classic uncoupler FCCP has an EC50 that is similar to that of PFOSA,
M570, and PFOSAA. FCCP can inhibit cellular proliferation and induce apoptosis in
cultured cells by causing the efflux of cytochrome c in mitochondria at levels near its
EC50 (Han, Kim et al. 2009; Han and Park 2009). Another classic uncoupler is 2, 4dinitrophenol, which has been associated with lipid peroxidation and oxidative stress in
isolated rat hepatocytes (Palmeira, Moreno et al. 1995) and with rapid weight loss and
hyperthermia in humans (Matsumoto, Furuhashi et al. 2008; Tewari, Ali et al. 2009).

Mode of action
42

The pKa values of the functional groups of the fluorinated compounds that were
included in this study are in the order of sulfonamides > carboxylic acids > sulfonic
acids. These pKa values also tend to increase with carbon chain length. The induction of
state 4 respiration by perfluorinated chemicals corresponded with increasing pKa values
(figure 4).
Of all of the PFAAs that were investigated in the current study, only PFOS and
PFDOA had a significantly stronger effect on state 3 respiration than PFOA. The
relatively potent effect that PFOS had on state 3 respiration indicates that its principle
toxicity toward mitochondria is mediated by inhibition of the respiratory chain or by
inhibition of ADP translocation. Although n-acetyl perfluorosulfonamides inhibit the
translocation of ADP through the adenine nucleotide translocator (ANT), PFOS does not
(O'Brien and Wallace 2004; O'Brien, Oliveira et al. 2008). As a result, it appears that
PFOS inhibits respiration by causing a nonspecific increase in membrane permeability
(Hu, Jones et al. 2003). This was evidenced in the current study by an initial induction of
state 4 respiration rates after exposure to PFOS.
In vivo studies have indicated that PFBS is less toxic and has far greater
elimination rates in rodents than PFAAs with longer carbon chains (Lau, Anitole et al.
2007). PFBS has a benign toxicological profile in Sprague-Dawley rats (Lieder, Chang et
al. 2009). The only observable physiological impact that PFBS caused in these animals
were minor histological changes to the kidney. The chain-length dependent inhibition of
fatty acid transporters and acyl-CoA synthetase correlates with the toxicological data that
suggest PFBA does not cause an important effect on lipid metabolism. Combined with
43

the modest effect that PFBS has on mitochondrial respiration, this evidence suggests that
PFBS does not pose a threat to mitochondrial dysfunction in humans, and that it may be a
viable alternative to longer-chain PFAAs for industrial purposes.
PFBA caused increased liver weights and delayed developmental characteristics
such as eye opening rates and the onset of puberty in mice (Das, Grey et al. 2008). PFBA
also induced hepatic effects in mice through activation of PPAR (Foreman, Chang et al.
2009), and the effect of PFAAs on PPAR was carbon-chain length dependent (Bjork
and Wallace 2009). Because peroxisome proliferation is more sensitive to PPAR in rats
than in humans (Ammerschlaeger, Beigel et al. 2004), human primary hepatocytes were
less responsive than rat primary hepatocytes to PFBA treatment (Bjork and Wallace
2009). PFBA is also more rapidly eliminated than longer-chain perfluorocarboxylic acids
in rodents and humans (Das, Grey et al. 2008; Weaver, Ehresman et al.). Together with
the current study, this data indicates that PFBA presents a lesser potential for human
toxicity than PFAAs with longer carbon chain lengths.
Because many perfluorinated chemicals are not as biologically prevalent as PFOA
or PFOS, their toxicological mechanisms have not been fully determined. This study
utilized model systems to determine whether other perfluorinated chemicals should also
be scrutinized. An advantage of the current study is that the respiration experiments were
performed in vitro on mitochondria isolated from livers of untreated rats. This allowed
the impact that perfluorinated chemicals have on mitochondrial respiration to be isolated,
and eliminated the consideration of other toxicological parameters such as the elimination
rates that are specific to individual perfluorinated compounds. Because perfluorinated
44

chemicals act directly on mitochondrial respiration, only the immediate effect of the
perfluorinated compounds on mitochondrial respiration was included in this study
(Starkov and Wallace 2002). However, the application of this study to human toxicity is
also limited because it does not include the downstream effects of perfluorinated
chemicals, such as the transactivation of PPAR or metabolic dysfunction, which may be
more important predictors of their toxicity. It should also be noted that PFAAs can
mediate PPAR-induced toxicity in rodents but not in humans (Ammerschlaeger, Beigel
et al. 2004), so they likely cause different long-term metabolic effects in rats than in
humans (Bjork and Wallace 2009). Furthermore, the relative exposure of mitochondria to
perfluorinated chemicals in vivo is dependent on their physiological distribution and
elimination rates. It is important to note that the EC50 of the most potent PFAA on
mitochondrial respiration, PFOSA, translates to a concentration of approximately 0.1
ppm, which compares to average human sera concentrations of approximately 0.1 ppb.
While mitochondrial respiration is only one toxicological parameter that is
impacted by perfluorinated chemicals, this study will complement other data that has
measured the relative impact of perfluorinated chemicals on peroxisome proliferation and
other toxicological parameters (Ohmori, Kudo et al. 2003; Kudo, Suzuki-Nakajima et al.
2006; Bjork and Wallace 2009). It will also help to determine the importance of carbon
chain length and functional group on mitochondrial respiration. Long-term toxicological
effects of perfluorinated chemicals on mitochondria continue to be investigated.
However, the current study is unique in that it provides a means by which the impact of

45

structurally different perfluorinated chemicals on mitochondrial respiration can be


directly compared.

46

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