Mitochondrial Permeability Transition as the Critical Target of N-Acetyl Perfluorooctane Sulfonamide Toxicity in Vitro

Timothy M. O'Brien and Kendall B. Wallace1

Department of Biochemistry and Molecular Biology, Toxicology Graduate Program, University of Minnesota School of Medicine, 1035 University Drive, Duluth, Minnesota 55812

Received June 1, 2004; accepted August 3, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Perfluorooctanyl compounds with active functional groups have been shown to disrupt mitochondrial bioenergetics by three distinct mechanisms: protonophoric uncoupling of mitochondrial respiration, induction of the mitochondrial permeability transition (MPT), or a nonselective increase in membrane permeability. The purpose of this investigation was to identify the initial target and specific sequence of events associated with the N-acetyl substituted perfluorooctanesulfonamides induced MPT. N-acetyl-perfluorooctanesulfonamide (FOSAA), N-ethyl-N-acetyl-perfluorooctanesulfonamide (N-Et FOSAA), perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS), and N-ethyl-N-(2-ethoxy)-perfluorooctanesulfonamide (N-Et FOSE) were added individually to liver mitochondria freshly isolated from Sprague-Dawley rats. Mitochondrial swelling and cytochrome c release were recorded spectrophotometrically, oxygen uptake was monitored with a Clark-type oxygen electrode, and reactive oxygen species (ROS) were monitored by dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence. FOSAA (45 µM) and N-Et FOSAA (7.5 µM) induced calcium-dependent mitochondrial swelling, the release of cytochrome c, inhibition of uncoupled mitochondrial respiration, and ROS generation, all of which were inhibited by cyclosporin-A (CsA). PFOA (200 µM) displayed slight CsA sensitive activity, but neither PFOS (10 µM) nor N-Et FOSE (70 µM) induced the MPT. Results of this investigation demonstrate two important findings: (1) MPT induction is specific to the N-acetyl substituted perfluorooctanesulfonamides and, (2) the sequence of events is initiated by induction of the MPT, which causes the release of cytochrome c as well as other cofactors leading to inhibition of respiration and ROS generation. The toxicity of N-acetyl perfluorooctanyl compounds may therefore reflect the mitochondrial dysfunction, which is compounded by the ensuing oxidative injury.

Key Words: mitochondria; permeability transition; PFOA; PFOS.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Perfluorooctanyl compounds such as perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), perfluorooctanesulfonamide (FOSA), and N-ethyl-N-acetyl-perfluorooctanesulfonamide (N-EtFOSAA) have surface tension reducing properties that have been used in numerous commercial applications (Kannan et al., 2002Go). N-EtFOSE is an intermediate in the synthesis of these perfluorinated compounds and other commercial products once widely used in oil and water repellant applications on both fabrics and papers (Olsen et al., 2004Go). PFOS, in addition to commercial use in surfactant applications, is an environmental and metabolic degradation product of certain N-alkyl-substituted perfluorooctanesulfonamide chemistries (Seacat et al., 2003Go). The high-energy fluorine-carbon bonds impart the physical and chemical stability that renders PFOS and PFOA resistant to hydrolysis, photolysis, microbial degradation and metabolism, and, consequently, cause them to be environmentally persistent (Giesy and Kannan, 2002Go; Shabalina et al., 1999Go; Starkov and Wallace, 2002Go). As an example PFOS and PFOA have been detected in birds, fish, and mammal tissues from the Canadian Artic, Baltic and Mediterranean Seas, and the Great Lakes of North America (Giesy and Kannan, 2002Go; Kannan et al., 2001Go, 2002Go; Martin et al., 2004Go). PFOS, PFOA, and N-EtFOSAA have also been identified in individual and pooled human serum samples from the United States general population (Hansen et al., 2001Go; Olsen et al., 2003aGo, 2004Go). The broad application, global distribution, and environmental persistence of these perfluorinated compounds has generated considerable concern regarding their potential toxicity (Renner, 2001Go).

The most prominent toxic effects of perfluorinated acids in rodents are altered lipid metabolism, hepatomegaly, and decreased body weight, suggesting a potential role for mitochondrial dysfunction in the underlying mechanism of toxicity. PFOA, FOSA, FOSAA, N-EtFOSAA, N-EtFOSE, and N-EtFOSA have been shown to disrupt mitochondrial bioenergetics in vitro by three different mechanisms: uncoupling mitochondria respiration, increasing nonspecific membrane permeability, and induction of calcium-dependent mitochondrial swelling (Keller et al., 1992Go; Panaretakis et al., 2001Go). The fact that uncoupling of respiration and induction of mitochondrial swelling occur at low concentrations suggest mitochondrial dysfunction may be an important factor in the pathogenesis caused by perfluorooctanyl compounds with active functional groups.

The mitochondrial permeability transition (MPT) has been identified as a mechanism by which perfluorinated carboxylic acids disrupt mitochondrial bioenergetics (Panaretakis et al., 2001Go; Starkov and Wallace, 2002Go). The MPT is a phenomenon whereby the exquisitely controlled permeability of the inner membrane is disrupted and mitochondria become nonselectively permeable to solutes up to 1.5 kD (Bernardi et al., 1992Go; Zoratti and Szabo, 1995Go). Associated with this is the rapid equilibration of solutes across the mitochondrial membranes leading to depolarization of membrane potential, osmotic swelling, and release of potential apoptogenic factors (Kroemer and Reed, 2000Go), including cytochrome c (Cai et al., 1998Go). It is plausible that the MPT-induced loss of mitochondrial function, release of cytochrome c, and the resultant ROS generation may account for the cell death associated with perfluorooctyl-mediated toxicity. The purpose of this investigation was to identify the initial target of PFOA and N-acetyl substituted perflorooctanesulfonamide induced mitochondrial dysfunction, and to fully characterize the sequential relationship between induction of the MPT, the release of cytochrome c, inhibition of mitochondrial respiration, and the generation of ROS. The compounds chosen for inclusion in this investigation are depicted in Table 1.


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TABLE 1 Structures of the Perfluorooctanes

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondria were isolated from liver of adult male Sprague-Dawley rats (200–300 g body weight) by differential centrifugation (Zhou and Wallace, 1999Go). Rats were purchased from Harlan Sprague-Dawley (Madison, WI) and acclimated in an AAALAC-accredited, climate-controlled animal-care facility for at least three days and fasted for 12–18 h prior to the experiment. Animals were euthanized by decapitation, and the liver was immediately excised, weighed, immersed in 40 ml of isolation medium (200 mM mannitol, 10 mM sucrose, 5 mM Hepes, 1 mM EGTA, pH 7.4) and minced with scissors. The tissue was rinsed in isolation medium until the supernatant appeared clear. The tissue suspension was then homogenized for 1 min in the same medium with a motor-driven Teflon-pestle Wheaton homogenizer. The homogenate was then centrifuged at 900 x g for 10 min and 4°C. Any floating debris was aspirated from the supernatant and the supernatant was collected and centrifuged at 9000 x g for 10 min and 4°C. The mitochondrial pellet was resuspended in 3 ml of washing medium (200 mM mannitol, 10 mM sucrose, 5 mM Hepes, pH 7.4) supplemented with fatty acid free bovine serum albumin (BSA, 1 mg/ml). The suspension was diluted to 40 ml with washing medium and centrifuged at 9000 x g for 10 min and 4°C. The resulting mitochondrial pellet was resuspended in washing medium to a final protein concentration of 90–110 mg/ml. Protein concentration was determined with the Bradford assay using BSA as a standard (Bradford, 1976Go).

The concentrations of the compounds tested were chosen based upon the results of previous studies. Panaretakis et al. (2001)Go treated human hepatoma HepG2 cells with PFOA at concentrations ranging from 150–500 µM. Starkov and Wallace (2002)Go established concentrations for all the perfluorinated sulfonamides at which they disrupted mitochondrial bioenergetics, and Luebker et al. (2002)Go established that 10 µM PFOS was a potent inhibitor of rat liver fatty acid binding protein in vitro. Using these results as background and confirmed by preliminary experiments, the following concentrations of perfluorinated compounds were chosen: 200 µM PFOA, 10 µM PFOS, 45 µM FOSAA, 70 µM N-EtFOSE, and 7.5 µM N-EtFOSAA.

Mitochondrial swelling. Changes in mitochondrial volume were estimated by changes in light scattering as monitored spectrophotometrically at 540 nm (Henry and Wallace, 1995Go; Starkov and Wallace, 2002Go). Freshly isolated mitochondria were suspended at 0.5 mg/ml in 200 mM sucrose, 10 mM Tris-MOPS, 1 mM KH2PO4, and 10 µM EGTA (pH 7.4) supplemented with 2 µM rotenone to prevent to backflow of electrons from ubiquinone to complex I when using succinate as a respiratory substrate and 1 µg/ml oligomycin to maintain a constant ADP/ATP ratio. The reaction was stirred continuously at 30°C for 20 min. The mitochondria were energized with 5 mM succinate for 2 min before adding 10–20 µM CaCl2. PFOA, FOSAA, N-EtFOSAA, PFOS, or N-EtFOSE was added 2 min later. Where indicated, 1 µM of the MPT inhibitor cyclosporin A (CsA) (Broekemeier et al., 1989Go) was added just prior to succinate. The criteria for establishing induction of the MPT was a decrease in light scattering over 20 min of at least 0.5 absorbance units which was inhibited completely by 1µM CsA.

Mitochondrial oxygen consumption and inhibition of uncoupled respiration. Mitochondria were suspended at a concentration of 0.5 mg/ml in 150 mM KCl, 5 mM KH2PO4, 5 mM Tris-MOPS (pH 7.4). The incubations were conducted at 28°C in a closed reaction chamber with constant stirring (Custodio et al., 1998Go; Starkov and Wallace, 2002Go) and oxygen concentration was monitored continuously with a Clark-type oxygen electrode. Chemical additions were made directly into the reaction chamber through a small portal. The mitochondria were energized with 5 mM each glutamate and malate for 1 min before adding 10 µM CaCl2. Nonphosphorylating, state-4 respiration was monitored for at least 2 min prior to adding 100 µM ADP to initiate state-3 respiration. Oxygen tension was monitored continuously until state-4 respiration resumed, at which point 40 µM 2,4-dinitrophenol was added to initiate uncoupled respiration, which was monitored for 30 s before adding PFOA, PFOS, FOSAA, N-EtFOSE, N-EtFOSAA, or high concentration (50 µM) CaCl2. Where indicated, 1 µM CsA was added just prior to glutamate/malate.

Cytochrome c release. The quantitative determination of cytochrome c released from isolated mitochondria was performed by measuring the Soret ({gamma}) peak for cytochrome c at 414 nm ({varepsilon} = 100 mM–1 cm–1), according to previously established methods (Appaix et al., 2000Go). Mitochondria were suspended at a concentration of 3 mg protein/ml in 1 ml of 150 mM KCl, 5 mM KH2PO4, 5 mM Tris-MOPS, (pH 7.4) supplemented with 10 µM CaCl2 and 5 mM each of glutamate and malate. Incubations were conducted for 30 min at 25°C in the presence of PFOA, PFOS, FOSAA, N-Et-FOSE, N-Et-FOSAA, or high concentration CaCl2. Where indicated, 1 µM CsA was added just prior to glutamate/malate. As a positive control, mitochondria were incubated in a hypotonic medium (60 mM KCl, 10 mM Hepes, pH 7.4) to induce maximum cytochrome c release. After incubation, mitochondria were centrifuged at 13,000 x g for 4 min, and the supernatant was immediately recovered by aspiration and cytochrome c measured spectrophotometrically. None of the reagents interfered with the spectrophotometric analysis.

ROS detection. Measurements of mitochondrial hydrogen peroxide were performed using the fluorescent probe dichlorodihydrofluorescein diacetate (H2DCFDA), in isolated rat liver mitochondria as previously described (McLennan and Degli Esposti, 2000Go). Briefly, mitochondria were incubated for 30 min at 28°C with H2DCFDA (4 nmol/mg) in the same medium as described for mitochondrial swelling. Once incorporated, H2DCFDA is deacylated by mitochondria esterases (McLennan and Degli Esposti, 2000Go). The deacylated product (H2DCF) is oxidized by H2O2 to the fluorescent DCF (McLennan and Degli Esposti, 2000Go). Afterwards, the suspension was again centrifuged at 8000 x g for 5 min to remove the nonincorporated probe. The mitochondrial pellet was re-suspended in washing medium to a final protein concentration of 70–80 mg/ml and stored on ice.

Mitochondria were then suspended at 0.5 mg/ml in 1 ml of respiration medium supplemented with 2 µM rotenone, 10–20 µM CaCl2, and 5 mM succinate. Incubations were conducted for 20 min at 30°C in the presence of PFOA, FOSAA, N-EtFOSAA, PFOS, N-EtFOSE, or 50 µM CaCl2. Where indicated, 1 µM CsA was added just prior to succinate. The DCF fluorescence was recorded continuously in a 24-well plate with a fluorescence multi-well plate reader with excitation and emission wavelengths at 485 and 538, respectively.

Reagents. All fluorochemical compounds were synthesized, characterized, and provided gratis by The 3 M Company, St. Paul, MN. Cyclosporin A was provided as a generous gift of Sandoz Pharmaceuticals (East Hanover, NJ), Ultra Pure sucrose was purchased from ICN Biomedicals, Inc. (Aurora, OH), H2DCFDA was purchased from Molecular Probes (Eugene, OR), and all other reagents were from Sigma-Aldrich (St. Louis, MO).

Statistical analysis. All experiments were repeated using freshly isolated hepatic mitochondria from at least three separate animals. The results were analyzed by two-way ANOVA and Tukey's post-hoc test, or by the Student's paired t-test (respiration data) depending upon the experimental design. A probability of p < 0.05 was used as the criterion for statistical significance.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+ is electrophoretically accumulated by mitochondria up to 1 µmol/mg protein via the near 200 mV electronegative transmembrane potential (Gunter and Gunter, 1994Go). However, there is a finite capacity which, if exceeded, triggers the mitochondria to undergo a dramatic change in permeability characteristics resulting in dissipation of the membrane potential and all trans-membrane chemical gradients leading to uncontrolled swelling. Collectively, this is referred to as induction of the mitochondrial permeability transition (MPT) (Bernardi, 1996Go; Zoratti and Szabo, 1995Go). Defining characteristics of the MPT are that it requires the accumulation of Ca2+ in the matrix and is inhibited by the cyclophilin D ligand CsA (Broekemeier et al., 1989Go; Zoratti and Szabo, 1995Go). In order to quantify calcium-loading capacity, which is considered to be a sensitive and specific indicator of mitochondrial competence and sensitivity to induction of the MPT (Starkov and Wallace, 2002Go; Zhou et al., 2001Go), mitochondria were titrated with concentrations of calcium ranging from 10–50 µM (data not shown). From this we established a concentration of 10 µM calcium (20 nmol/mg protein) as an amount that is below the calcium loading capacity of control mitochondria, yet sufficient to support induction of the MPT in response to selected inducing agents.

Figure 1 demonstrates the typical experimental run for testing the effects of fluorochemical test compounds on mitochondrial swelling. In each case, the rate and extent of mitochondrial swelling depended on the dose and identity of the compound tested. For example, 7.5 µM N-EtFOSAA and 45 µM FOSAA caused a profound decrease in mitochondrial light scattering that was inhibited by 1 µM CsA. In contrast, 200 µM PFOA induced a very limited mitochondrial swelling that was inhibited by CsA. However, at higher CaCl2 concentrations, 200 µM PFOA did not change the rate or amplitude of light scattering beyond that observed with controls, suggesting that PFOA does not act as a classical MPT inducer (data not shown). Likewise, neither 10 µM PFOS nor 70 µM N-EtFOSE induced mitochondrial swelling. This data provides strong evidence that the perfluorooctanesulfonamides with carboxylic acid functionality induce the MPT. In contrast, PFOA displays slight CsA sensitive swelling, but the high concentration suggests that PFOA is, at best, a very weak inducer of the MPT. The noncarboxylated compounds PFOS and N-EtFOSE did not induce the MPT.



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FIG. 1. Mitochondrial swelling by (A) 7.5 µM N-Et-FOSAA, (B) 45 µM FOSAA, (C) 200 µM PFOA, and (D) 10 µM PFOS and 70 µM N-Et-FOSE. Rat liver mitochondria (0.5 mg protein/ml) were incubated at 30°C in 200 mM sucrose, 10 mM MOPS, 1 mM KH2PO4, 10 µM EGTA in the presence of 2 µM rotenone, 5 mM succinate, 2 µg/ml oligomycin, 10 µM Ca2+ plus the selected perfluorinated compound at the indicated concentration in the presence and absence of 1 µM cyclosporin A (CsA). Perfluorinated compounds were dissolved in DMSO. Controls were treated with 10 µM Ca2+ as well as a dose of DMSO equivalent to the volume of the corresponding treatment. Mitochondrial swelling was monitored by light scattering at 540 nm. Traces are representative of 4–8 repetitions, each using separate mitochondrial preparations.

 
The relationship between treatment with the test compounds and cytochrome c release is illustrated in Figure 2, where the amount of cytochrome c released by mitochondria exposed to 50 µM calcium, PFOA, FOSAA, N-EtFOSAA, PFOS, or N-EtFOSE, in the presence or absence of CsA is compared to untreated mitochondria (10 µM CaCl2) and mitochondria exposed to a hypotonic media as a positive control. The data reveal that treatment with 45 µM FOSAA, 7.5 µM N-EtFOSAA, or 50 µM calcium resulted in the release of cytochrome c, which was inhibited by CsA, suggesting that cytochrome c release is the result of induction of the MPT. Treatment with 200 µM PFOA resulted in significant increase in the release of cytochrome c compared to untreated mitochondria. However, unlike N-acetyl perfluorooctanesulfonamides, PFOA induced cytochrome c release was not inhibited by cyclosporin A. Neither 10 µM PFOS nor 70 µM N-EtFOSE induced cytochrome c release compared to control.



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FIG. 2. Cytochrome c release: Freshly isolated rat liver mitochondria (3 mg protein) were incubated in 1 ml buffer (150 mM KCl, 5 mM H2KPO4, 5 mM Tris [pH 7.4]) at a final concentration of 3.0 mg/ml in the presence of the test compounds and in the presence or absence of 1 µM cyclosporin A (CsA) supplemented with glutamate/malate and 10 µM CaCl2 for 30 min at 25°C. The hypotonic buffer, 60 mM KCl, 10 mM Hepes was used as a positive control. Cytochrome c concentration was measured at 414 nm ({varepsilon} = 100 mM–1 cm–1). Bars represent the mean ± SEM for seven individual mitochondrial preparations. *Statistically significant difference compared to untreated control as determined by two-way ANOVA and Tukey's post-hoc test (p < 0.05). {dagger}Statistically significant difference compared to treatment in the absence of CsA as determined by two-way ANOVA and Tukey's post-hoc test (p < 0.05).

 
The implication of exposure to the fluorochemicals with carboxylate functionality is that release of cytochrome c results in the inhibition of mitochondrial respiration by inhibiting electron transport to cytochrome oxidase (COX). To test this hypothesis we assessed the effect of the fluorochemical compounds on uncoupled mitochondrial respiration. Maximum respiration rate was achieved by the addition of the mitochondrial uncoupler 2,4-dinitrophenol (40 µM DNP) and then the specified test compound was added approximately 30 s later. It is well established that sufficiently high calcium concentration will completely and irreversibly induce the MPT. Throughout this study, 50 µM calcium was used as a positive treatment control. Figure 3 demonstrates that mitochondrial respiration was significantly inhibited by 50 µM calcium, a concentration that caused mitochondrial swelling and cytochrome c release (Fig. 2). CsA prevented inhibition of uncoupled respiration by 50 µM calcium, which implicates that MPT release of cytochrome c blocks respiration. Forty-five µM FOSAA, and 7.5 µM N-EtFOSAA, but not by 200 µM PFOA, 10 µM PFOS or 70 µM N-EtFOSE, also inhibited uncoupled respiration. In all cases inhibition of uncoupled mitochondrial respiration by N-acetyl perfluorooctanesulfonamides was prevented by CsA, strongly implicating induction of the MPT as the precipitating event.



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FIG. 3. Uncoupled respiration: Freshly isolated rat liver mitochondria were incubated at 0.5 mg/ml and 28°C in 1 ml buffer (150 mM KCl 5 mM H2KPO4, 5 mM Tris [pH 7.4]) supplemented with glutamate/malate, 10 µM Ca2+, 100 µM ADP, and after the resumption of resting respiration, dinitrophenol (DNP), plus the specified compound in the presence or absence of 1 µM cyclosporin A (CsA) were added. Each bar represents the mean ± SEM for 3–8 individual mitochondrial preparations. The control bar represents the mean of all 28 controls. *Statistically significant difference compared to the paired uncoupled control absent the test compound as determined by the paired Student's t-test (p < 0.05).

 
In order to investigate whether the release of cytochrome c and inhibited respiration caused by the perfluorooctanesulfonamides with carboxylic acid functionality results in mitochondrial ROS generation, we monitored ROS production with the H2O2 specific fluorescent probe H2DCFDA (Fig. 4). The results demonstrate that there was a significant increase in the rate of ROS production by high (50 µM) calcium, FOSAA, and N-EtFOSAA treated mitochondria versus the untreated control (10 µM calcium). CsA blocked the stimulation of ROS production in each case, although FOSAA + CsA was not statistically different than FOSAA alone because of the large experimental variability in the data. There was a significant difference in ROS production by PFOA compared to PFOA + CsA treated, but not compared to control. Neither PFOS nor N-EtFOSE stimulated ROS generation compared to control.



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FIG. 4. Free radical generation: Freshly isolated rat liver mitochondria were incubated in sucrose buffer (200 mM sucrose, 10 mM MOPS, 1 mM KH2PO4, 10 µM EGTA) with 4-nmol/mg protein H2DCFDA at 30°C for 30 min. The suspension was centrifuged and the mitochondrial pellet resuspended at 0.5 mg/ml in 1 ml of buffer (150 mM KCl, 5 mM H2KPO4, 5 mM Tris [pH 7.4]) supplemented with 10 µM Ca2+ and the appropriate concentration of test compounds in the presence or absence of 1µM cyclosporin A. High calcium (50 µM) was used as a treatment control. The test compounds were dissolved in 100% EtOH. Each untreated control was supplemented with 10 µM Ca2+, and contained equivalent volume of EtOH as the matching treatment. Each bar represents the mean rate of change in fluorescent units during the first 10 min ± SEM for 4–5 individual mitochondrial preparations. The control bar represents the mean of all 28 controls. *Statistically significant difference compared with the controls as determined by two-way ANOVA and Tukey's post-hoc test (p < 0.05). {dagger}Statistically significant difference compared with treatment in the absence of CsA as determined by two-way ANOVA and Tukey's post-hoc test (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mitochondrial permeability transition represents a non-selective increase in the permeability of the inner mitochondrial membrane to solutes up to 1.5 kD in size (Bernardi, 1996Go). It is characterized by dependence on matrix Ca2+, alkalization of the matrix and inhibition by CsA. Ca2+ can itself induce the MPT, but most often the MPT is a result of Ca2+ acting in conjunction with various ‘inducing agents’ such as fatty acids or oxidants (Petronilli et al., 1994bGo). The ultimate effect of the MPT is dissipation of mitochondrial membrane potential and inhibition of oxidative phosphorylation (Zoratti and Szabo, 1995Go) leading to large amplitude swelling and the release of cytochrome c and a variety of other apoptogenic factors.

The present investigation was designed to identify critical targets and the sequence of ensuing events associated with perfluorinated carboxylic acid-induced disruption of mitochondrial bioenergetics in vitro. Carboxylic acids of various structures are well known inducers of the MPT in vitro (Custodio et al., 1998Go; Palmeira et al., 2000Go; Schonfeld and Bohnensack, 1997Go; Starkov and Wallace, 2002Go; Zoratti and Szabo, 1995Go), suggesting that the key feature is the carboxylic acid functionality. The N-acetyl perfluorooctanesulfonamides, FOSAA and N-EtFOSAA, produced results that were consistent with previous observations with carboxylic acids as demonstrated by calcium-dependent swelling that was inhibited by CsA. In contrast, the non-carboxylated pefluorooctanesulfonamides, FOSA and N-EtFOSA did not induce the MPT. At best, PFOA may be considered a very weak inducer of the MPT. The slight mitochondrial swelling observed for PFOA was not entirely unexpected considering that as a surfactant; PFOA at these concentrations (200 µM) may have a detergent-like effect on mitochondrial membranes. Although PFOA exhibits some effects consistent with MPT induction, not all are inhibited by CsA; and thus, PFOA does not display the classic effects of an MPT inducer. In contrast the N-acetyl perfluorooctanesulfonamides are potent MPT inducers.

Results with the N-acetyl perfluorooctanesulfonamides indicate that the initial step in the disruption of mitochondrial bioenergetics in vitro is induction of the MPT, which then causes the release of cytochrome c as well as other cofactors leading to inhibition of respiration and generation of ROS. Although oxidative stress is widely invoked in regulating the MPT (Nieminen et al., 1997Go; Petronilli et al., 1994aGo), in this case ROS is the result and not the cause of induction of the MPT as indicated by the following observations: (1) the N-acetylated perfluorooctanesulfonamides do not spontaneously generate ROS (data not shown); (2) the release of cytochrome c and inhibition of uncoupled respiration is prevented by CsA; and (3) ROS generation is prevented by CsA. The fact that all of these events occur with the N-acetyl perfluorooctanesulfonamides but not the perfluorinated carboxylate PFOA, the noncarboxylated sulfonamides FOSA, N-EtFOSA (Starkov and Wallace, 2002Go), and N-EtFOSE, or the sulfonic acid PFOS implicates the necessity of N-acetyl functional group for potent MPT induction.

Treatment of rodents with PFOS or N-EtFOSE demonstrates a toxicological profile resembling that of PFOA (Berthiaume and Wallace, 2002Go; Haughom and Spydevold, 1992Go; Ikeda et al., 1985Go; Keller et al., 1992Go; Luebker et al., 2002Go; Pastoor et al., 1987Go; Seacat et al., 2002Go). In vitro PFOS or N-EtFOSE did not induce calcium-dependent mitochondrial swelling, cytochrome c release, inhibition respiration, or stimulation of ROS generation. This does not, however, discount the MPT in the pathogenesis of N-EtFOSE, as N-acetates may be prominent oxygenated intermediates of N-EtFOSE metabolism, with PFOS as the terminal product (Seacat et al., 2003Go).

PFOS has generated considerable interest recently because of its environmental prevalence and persistence. It has been detected in a variety of species of wildlife worldwide with mean liver concentrations as high as 1.7 ppm in top predators such as polar bears (Martin et al., 2004Go). Exposure of the general human population is demonstrated by the detection of PFOS in banked serum at concentrations of 0.03–0.175 ppm (Hansen et al., 2001Go; Olsen et al., 2003bGo, 2004Go) with occupational exposures of fluorochemical production workers reported at 0.04–12.70 ppm serum PFOS (Olsen, 2003aGo). Since PFOS has been suggested to be the terminal metabolite of the N-alkyl-perfluorooctanesulfonamides in vivo (Renner, 2001Go; Seacat et al., 2002Go; Thibodeaux et al., 2003Go), the detection of PFOS in human or environmental monitoring studies may reflect either direct exposure to PFOS itself or exposures to more complex structural analogs. Comparing detected serum concentrations of PFOS to the concentrations of FOSAA (~25 ppm) and N-EtFOSAA (~3.7 ppm) used in this study supports the plausibility that mitochondrial dysfunction may represent a significant component in the pathogenesis of the perfluorooctanoyl compounds.

In conclusion, we demonstrate that N-acetyl perfluorooctanesulfonamides, FOSAA, and N-EtFOSAA, induce the CsA-sensitive mitochondrial permeability transition in vitro. The mechanistic sequence is initiated by induction of the MPT and mitochondrial swelling, resulting in the release of cytochrome c, which inhibits mitochondrial respiration leading to increased rates of ROS production. CsA inhibits all of these events, demonstrating the critical role of the MPT in initiating mitochondrial dysfunction caused by the N-acetyl perfluorooctanesulfonamides. In contrast, although PFOA caused membrane disruption, the MPT is not a significant target of PFOA toxicity. The fact that induction of the MPT in vitro by the N-acetyl perfuorooctanesulfonamides was observed at concentrations approximating those of the terminal metabolite PFOS detected in serum supports the possible significance of mitochondrial dysfunction in the pathogenesis of N-acetyl perfluorooctanesulfonamides following environmental or occupational exposures.


    ACKNOWLEDGMENTS
 
This work was supported by a generous grant from the 3 M Company. The authors would also like to thank James A. Bjork, Jessica M. Berthiaume, and Kaleb C. Lund for their technical support and insight, and Dr. John L. Butenhoff for his expert review of this manuscript.


    NOTES
 

1 To whom correspondence should be addressed. Fax: (218) 726-8014. E-mail: kwallace{at}d.umn.edu.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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