Department of Biochemistry and Molecular Biology, University of Minnesota School of Medicine, 10 University Drive, Duluth, Minnesota
Received September 7, 2001; accepted December 19, 2001
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ABSTRACT |
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Key Words: mitochondria; perfluoroalkanes; structure-activity; peroxisome proliferator.
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INTRODUCTION |
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The wasting syndrome, or cachexia, observed with perfluorinated acid exposures in experimental animals is a dose-limiting event and attributed to a metabolic disorder at the cellular level. Biochemical manifestations of PFOA, PFDA, and PFOS exposure are indicative of altered lipid metabolism and include a lowering of serum triglycerides and cholesterol and the accumulation of lipid droplets in liver cells (Haughom and Spydevold, 1992; Pastoor et al., 1987
; Sohlenius et al., 1993
; Vahden Heuvel, 1996
). Enzyme analysis reveals an inhibition of HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase activities in livers of rats fed either PFOA or PFOS (Sohlenius et al., 1993
). Histomorphology reveals an expansion of peroxisomal bodies that is accompanied by the stimulation of peroxisome-related enzyme activities such as acyl CoA oxidase and catalase (Pastoor et al., 1987
; Permadi et al., 1992
, 1993
; Sohlenius et al., 1992
; Vahden Heuvel, 1996
; Van Rafelghem et al., 1987
). From a structure-activity standpoint, longer-chain perfluorinated fatty acids are more potent than short-chain acids (Goecke-Flora and Reo, 1996
). In view of these observations, perfluorinated acids have been categorized as peroxisome proliferators and hypothesized to act as structural mimics of fatty acids, thereby being competitive inhibitors of mitochondrial ß-oxidation. The reported increase in mitochondrial protein content in liver of exposed rats may represent either a condensation and change in buoyancy of individual mitochondria (Permadi et al., 1993
) or a compensatory mitochondrial biogenesis. Regardless, the mitochondrion has emerged as a primary intracellular target for perfluorinated acid-induced hepatotoxicity.
In addition to the expansion of the mitochondrial fraction within individual hepatocytes (Pastoor et al., 1987; Permadi et al., 1992
, 1993
; Sohlenius et al., 1992
), a number of perfluorinated acids have been reported to uncouple mitochondrial respiration. For example, PFOA and PFDA stimulate state 4 respiration and uncouple state 3 respiration in isolated rat liver mitochondria (Keller et al., 1992
; Langley, 1990
). This uncoupling of mitochondrial respiration has also been demonstrated for isolated perfused liver from rats exposed in vivo to PFOA in their diet (Keller et al., 1992
). Schnellmann and Manning (1990) demonstrated that perfluorooctane sulfonamide (FOSA) is a protonophoric uncoupler of oxidative phosphorylation in isolated rat kidney mitochondria and suggested that this may account for the nephrotoxicity caused by the insecticide sulfluramid (N-ethyl-FOSA).
Based on the results to date, it can be proposed that the critical molecular event that mediates perfluorinated carboxylic and sulfonate-induced interference with hepatic lipid metabolism and stimulation peroxisome proliferation is that they act as protonophoric uncouplers of mitochondrial respiration. On a molecular basis, this requires certain physical chemical features of the individual compounds: They must be sufficiently hydrophobic to traverse the inner mitochondrial membrane, possess an ionizable atom with a pKa in the range of 57, and contain a fairly strong electron-withdrawing moiety (Wallace and Starkov, 2000). The purpose of the current investigation was to more thoroughly describe the specific mechanisms by which the various perfluorooctanes interfere with mitochondrial bioenergetics and to identify the key structural/physical chemical features responsible for these activities. Structures of the compounds selected for this investigation are illustrated in Table 1
.
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MATERIALS AND METHODS |
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Mitochondrial membrane potential () was estimated from TPP+ ion distribution measured with a TPP+- selective electrode constructed according to Kamo et al. (Kamo et al., 1979
). Mitochondrial membrane potential was calculated as described elsewhere (Custodio et al., 1998
; Rottenberg, 1984
). The rate of oxygen consumption by mitochondria was measured with a Clark-type oxygen electrode. Both the mitochondrial membrane potential and the respiration rate were recorded simultaneously using a multichannel incubation chamber equipped with a magnetic stirrer. The volume of the chamber was 1.8 ml. All experiments were performed at room temperature (25°C). The TPP+-sensitive electrode was calibrated by sequential additions of known amounts of TPP+Cl- before the addition of mitochondria (Fig. 1
). Preliminary experiments were performed to ensure that the ethanol, pH, or the compounds themselves did not interfere with the TPP+ or oxygen electrodes. Respiration rates were calculated assuming the initial oxygen concentration to be 240 µM. Protein concentration was determined by the Bradford assay (Bradford, 1976
) using BSA as a standard.
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Reagents.
All perfluorinated compounds were synthesized, characterized, and provided gratis by The 3M Company, St. Paul, MN. Ultra Pure sucrose was purchased from ICN Biomedicals, Inc. (www.icnbiomed.com), and all other reagents were from Sigma-Aldrich (www.sigma-aldrich.com). BSA was essentially fatty acidfree.
Statistical analysis.
All experiments were repeated at least three times using freshly isolated hepatic mitochondria from separate animals for each experimental repetition. The results were analyzed by the Student's paired t-test, using a probability of p < 0.05 as the criterion for statistical significance.
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RESULTS |
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Additional experiments were then performed to investigate the specific molecular mechanism by which each of these agents interfere with mitochondrial respiration. The first to be studied was the very potent uncoupler FOSA. Figure 2 illustrates the results of an experiment wherein both mitochondrial membrane potential and state 4 respiration were titrated with successive additions of FOSA. Each successive addition of FOSA caused a progressive depolarization of membrane potential and stimulation of state 4 respiration. The fact that this mimics very closely the response observed for agents such as 2,4-dinitrophenol (DNP) is highly suggestive of a protonophoric uncoupling effect, which is precisely what was reported by Schnellmann and Manning (Schnellmann and Manning, 1990
). Similar responses to increasing concentrations of the secondary amides N-EtFOSA and FOSAA were also observed (data not shown), suggesting that they too might be classified as protonophoric uncouplers. To help verify whether these three compounds elicit an uncoupling effect comparable to DNP, the data were analyzed in terms of quantifying the dependence of state 4 respiration on membrane potential (Fig. 3
). As would be expected, depolarization of mitochondrial membrane potential caused by successive additions of DNP was associated with proportionate increases in the rate of respiration. This linear relationship held true over a 50-mV range of membrane potentials, which indicates that none of these compounds directly inhibit the mitochondrial electron transport chain. The fact that similar relationships for membrane potentialdependent mitochondrial respiration existed for FOSA, N-EtFOSA, and FOSAA suggests that all three agents are DNP-like protonophoric uncouplers of mitochondrial respiration (Fig. 3
). The fact that they share the characteristic of being primary or secondary amides suggests that it is the protonated nitrogen atom that is key to this mechanism of uncoupling of mitochondrial respiration by the perfluorooctanesulfonamides. PFOA, PFOS, N-EtFOSE, and N-EtFOSAA did not demonstrate a similar linear relationship between respiration rate and membrane potential.
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Further experiments with the agents that were found not to be specific uncouplers of mitochondrial respiration revealed that at sufficiently high concentrations, PFOA, PFOS, and N-EtFOSE caused a small increase in resting respiration rate and slightly decreased the membrane potential, with no effect on the phosphorylating respiration or on the coupling efficiency of mitochondria. This might be tentatively attributed to induction of a slight increase in intrinsic proton leak of the mitochondrial inner membrane due to changes in its fluidity, which is a surfactant-like effect of these agents. The basis for the differential effect on states 3 and 4 respiration is that the intrinsic proton leak is membrane potential-dependent and thus greatest during state 4 when membrane potential is greatest. Adding ADP to stimulate state 3 respiration causes a transient depolarization of membrane potential (Fig. 1), thereby reducing protonmotive force responsible for the proton leak (Nicholls 1974a
,b
; 1997
). This reduction in protonmotive force was reflected by the decrease in resting (state 4) membrane potential observed with these three compounds (data not shown).
A particularly interesting observation was that at concentrations greater than 25 µM, the di-substituted sulfonamide N-EtFOSAA caused strong inhibition of state 3 respiration. Figure 5 illustrates this effect, where it is shown that adding 50 µM N-EtFOSAA inhibited state 3 respiration by 50%. The fact that N-EtFOSAA inhibited both succinate- and glutamate/malate-supported respiration suggests that N-EtFOSAA does not directly inhibit complex I or complex II of the respiratory chain. Adding DNP at a concentration that completely uncouples mitochondrial oxidative phosphorylation did not stimulate respiration beyond that recorded for N-EtFOSAA by itself. The fact that adding cytochrome c back to the reaction chamber restored respiration indicates that the inhibition by N-EtFOSAA is due to its causing the release of mitochondrial cytochrome c. Adding the cytochrome back to the reaction restored cytochrome oxidase activity (complex IV) and reestablished respiratory capability to the mitochondria. Such an effect suggests that N-EtFOSAA may be acting as an inducer of the mitochondrial permeability transition pore (MPTP).
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DISCUSSION |
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The data presented herein indicate that at sufficiently high concentrations, all of the tested perfluorinated octanyl acids and their derivatives are able to increase the nonselective permeability of mitochondrial membranes. This should not be surprising in view of the intended use of many of these agents as surfactants and surface-active water repellants. This is particularly true for PFOA and PFOS, which demonstrated no other specific effects on mitochondrial bioenergetics. Whether these effects have any physiological relevance remains to be determined and relies on the accumulation of the individual perfluorochemical in the targeted membrane at concentrations sufficient to interfere with membrane structure and fluidity.
Besides this general, nonspecific effect observed at high concentrations, selected perfluorooctanes caused fairly specific and sometimes highly potent effects on mitochondrial bioenergetics. One of the more common effects shared by the primary and secondary amides is the uncoupling of rat liver mitochondrial oxidative phosphorylation, apparently via a protonophoric mechanism similar to that caused by DNP. This same effect has been previously reported for PFOA and PFDA, but at much higher concentrations (150 µM0.5 mM; Keller et al., 1992; Langley, 1990
). We found that the ionizable perfluorooctanesulfonamides (FOSA, FOSAA, and N-EtFOSA) elicit a strong uncoupling of mitochondrial oxidative phosphorylation at concentrations of 550 µM. Such an effect is evident from the stimulation of state 4 respiration combined with the linear relationship between respiration rate and membrane potential. Schnellmann and Manning (1990) demonstrated specific protonophoric uncoupling of rabbit renal cortical mitochondria with low concentrations of FOSA (125 µM) and suggested that this might mediate the cytotoxicity of the insecticide sulfluramid (N-EtFOSA), which is metabolized in part to FOSA. Similar mechanisms have been implicated for the herbicide perfluidone (1, 1, 1-trifluoro-N-[2-methyl-4-(phenylsulphonyl)-phenyl]methanesulphonamide, which required only 5 µM to cause a doubling of state 4 respiration and a 50% decrease in RCR (Olorunsogo and Malomo, 1985
; Olorunsogo et al., 1985
). It is suggested that the ionizable amide, with a favorable pKa, shuttles protons back into the mitochondrial matrix, thereby dissipating the protonmotive force generated by the electron transport chain. The fully substituted amides N-EtFOSE and N-EtFOSAA, which lack the protonated amide, were found not to be uncouplers of mitochondrial respiration. Of particular concern is the striking potency of FOSA in uncoupling rat liver mitochondrial respiration. As illustrated in Figure 4
, FOSA is approximately five times more potent than DNP in stimulating state 4 respiration in isolated rat liver mitochondria. As little as 0.5 µM FOSA caused significant stimulation of mitochondrial respiration. This is of the same order of potency as FCCP, which is one of the most potent uncouplers of mitochondrial oxidative phosphorylation currently known. This concentration of FOSA translates to 0.25 ppm, a concentration that under certain conditions could conceivably occur from occupational or environmental exposures.
The other interesting finding is that high concentrations of FOSAA and N-EtFOSAA inhibit mitochondrial respiration by causing the release of cytochrome c from the inner mitochondrial membrane, thereby inhibiting the activity of cytochrome oxidase (COX). Replenishing with exogenous cytochrome c restored both COX activity and respiration rate. The basis for this is that both compounds induce what is known as the mitochondrial permeability transition (MPT), a phenomenon whereby the exquisitely controlled permeability of the inner mitochondrial membrane is lost and the mitochondria become nonselectively permeable to solutes of up to 1.5 kD (Bernardi et al., 1999; Petronilli et al., 1994
; Zoratti and Szabo, 1995
). Associated with this is the rapid equilibration of solutes across the mitochondrial membranes, leading to depolarization of the membrane potential and osmotic swelling. This, in turn, is proposed to cause physical disruption of the outer mitochondrial membrane to permit the release of cytochrome c and other apoptogenic factors that reside in the intermembrane space (Kroemer and Reed, 2000
). It is the accompanying inhibition of ATP synthesis and/or release of apoptogenic factors that has been the basis for implicating induction of the MPT in numerous and assorted cytopathies, including both chemical-induced as well as ischemia/reperfusion injuries (Lemasters et al., 1998
; Wallace et al., 1997
). Whether induction of the MPT occurs in response to exposures to the perfluorooctanesulfonamides in vivo and its role in mediating any ensuing metabolic dysfunction or tissue injury has yet to be defined.
In summary, the perfluorooctanes were found to elicit three basic effects on mitochondrial bioenergetics. All of the compounds at sufficiently high concentrations caused a nonspecific increase in ion permeability of the mitochondrial membrane, an effect that resembles that caused by membrane detergents. At much lower concentrations, the ionizable amides caused a specific and potent uncoupling of mitochondrial oxidative phosphorylation. In the case of FOSA, the potency compares with some of the most potent uncouplers of mitochondrial respiration known to date. The third mechanism by which selected perfluorooctanesulfonamides interfere with mitochondrial bioenergetics is by inducing the MPT, leading to inhibition of ATP synthesis and release of apoptogenic factors. In view of the primary role of mitochondria in glucose, fatty acid, and cholesterol metabolism in the cell, it is possible that any one of these effects could account for the metabolic disorders and cachexia observed in conjunction with perfluorooctane exposure to rats in vivo. Whether mitochondrial dysfunction is a factor in assessing human health risks associated with perfluorooctanyl exposure depends on whether similar effects are manifested in vivo and on the absence of remarkable species-related differences in sensitivity of the response.
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ACKNOWLEDGMENTS |
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NOTES |
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