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
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ABSTRACT |
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Key Words: mitochondria; permeability transition; PFOA; PFOS.
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INTRODUCTION |
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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., 1992; Panaretakis et al., 2001
). 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., 2001; Starkov and Wallace, 2002
). 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., 1992
; Zoratti and Szabo, 1995
). 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, 2000
), including cytochrome c (Cai et al., 1998
). 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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MATERIALS AND METHODS |
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The concentrations of the compounds tested were chosen based upon the results of previous studies. Panaretakis et al. (2001) treated human hepatoma HepG2 cells with PFOA at concentrations ranging from 150500 µM. Starkov and Wallace (2002)
established concentrations for all the perfluorinated sulfonamides at which they disrupted mitochondrial bioenergetics, and Luebker et al. (2002)
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, 1995; Starkov and Wallace, 2002
). 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 1020 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., 1989
) 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., 1998; Starkov and Wallace, 2002
) 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 () peak for cytochrome c at 414 nm (
= 100 mM1 cm1), according to previously established methods (Appaix et al., 2000
). 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, 2000). 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, 2000
). The deacylated product (H2DCF) is oxidized by H2O2 to the fluorescent DCF (McLennan and Degli Esposti, 2000
). 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 7080 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, 1020 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.
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RESULTS |
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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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DISCUSSION |
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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., 1998; Palmeira et al., 2000
; Schonfeld and Bohnensack, 1997
; Starkov and Wallace, 2002
; Zoratti and Szabo, 1995
), 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., 1997; Petronilli et al., 1994a
), 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, 2002
), 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, 2002; Haughom and Spydevold, 1992
; Ikeda et al., 1985
; Keller et al., 1992
; Luebker et al., 2002
; Pastoor et al., 1987
; Seacat et al., 2002
). 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., 2003
).
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., 2004). Exposure of the general human population is demonstrated by the detection of PFOS in banked serum at concentrations of 0.030.175 ppm (Hansen et al., 2001
; Olsen et al., 2003b
, 2004
) with occupational exposures of fluorochemical production workers reported at 0.0412.70 ppm serum PFOS (Olsen, 2003a
). Since PFOS has been suggested to be the terminal metabolite of the N-alkyl-perfluorooctanesulfonamides in vivo (Renner, 2001
; Seacat et al., 2002
; Thibodeaux et al., 2003
), 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.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (218) 726-8014. E-mail: kwallace{at}d.umn.edu.
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