Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effects of perfluoro‐n‐decanoic acid on the respiratory activity of isolated rat liver mitochondria Albert E. Langley To cite this article: Albert E. Langley (1990) Effects of perfluoro‐n‐decanoic acid on the respiratory activity of isolated rat liver mitochondria, Journal of Toxicology and Environmental Health, 29:3, 329-336, DOI: 10.1080/15287399009531395 To link to this article: http://dx.doi.org/10.1080/15287399009531395
Published online: 20 Oct 2009.
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Citing articles: 16 View citing articles
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Date: 06 November 2015, At: 12:31
EFFECTS OF PERFLUORO-n-DECANOIC ACID ON THE RESPIRATORY ACTIVITY OF ISOLATED RAT LIVER MITOCHONDRIA Albert E. Langley
Downloaded by [University of Florida] at 12:31 06 November 2015
Department of Pharmacology and Toxicology, Wright State University, School of Medicine, Dayton, Ohio
The toxic responses of rats to a single dose of perfluorodecanoic acid (PFDA) include reduced food consumption, severe body weight loss ("wasting syndrome"), and hypothermia. Recent studies have suggested that some of these effects may be due to alterations of basic metabolic processes in animals treated with PFDA. In order to test this hypothesis the effects of PFDA on respiratory activity of isolated rat liver mitochondria were examined. PFDA concentrations up to 87.5 µg/ml produced a linear increase in oxygen consumption during state 4 (nonphosphorylating) respiration. This suggested uncoupling of electron transport and oxidative phosphorylation was supported by the observation that PFDA released mitochondrial state 3 respiration from inhibition by oligomycin and stimulated latent ATPase activity. Concentrations of PFDA greater than 87.5 µg/ml produced progressively less increase in state 4 oxygen consumption, and a single high concentrations (150 µg/ml) completely inhibited state 3 respiration and prevented the uncoupling effect of 2,4-dinitrophenol. These observations suggest that in addition to uncoupling electron transport and oxidative phosphorylation PFDA may affect other energy-transducing functions of liver mitochondria such as inhibiting electron transport. These effects on mitochondrial respiration may help to explain the "wasting syndrome" characteristic of PFDA toxicity.
INTRODUCTION Perfluoro-n-decanoic acid (PFDA) is a member of a family of perfluorinated carboxylic acids that have found widespread commercial applications as lubricants, surfactants, wetting agents, corrosion inhibitors, and gloss enhancers of commercial aqueous emulsion type floor waxes (Bryce, 1964). Perfluorinated fatty acid surfactants have also been used to impart oil and water repellancy to cloth, leather, and paper. They are found in aqueous film-forming foam fire extinguishants (Shinoda and Nomura, 1980). In rats a single dose of PFDA near the LD50 has been reported to produce hypophagia and severe weight loss (Olson and Andersen, 1983; This work was supported by a grant from the Air Force Office of Scientific Research, AFOSR82-0264.
329 Journal of Toxicology and Environmental Health, 29:329-336, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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Langley and Pilcher, 1985), bradycardia, hypothermia, and decreased serum thyroid hormone levels (Langley and Pilcher, 1985). Serum thyroxine levels were significantly lower than control levels as early as 12 h after a single 75 mg/kg dose of PFDA, and were maximally reduced within 48 h (Langley and Pilcher, 1985). These data indicated that decreases in serum thyroid hormone levels were one of the earliest responses to a single injection of PFDA. Consequently rats were supplemented with thyroxine in an effort to prevent the toxic effects of PFDA. Supplementing with 200 ^g/kg-d thyroxine prevented PFDA-induced hypophagia but was ineffective at preventing the "wasting syndrome" and hypothermia (Gutshall et al., 1988). These observations coupled with the reports that PFDA-treated rats lost significantly greater weight than pair-fed controls (Langley and Pilcher, 1985; Olsen and Andersen, 1983; Van Rafelghem et al., 1988) suggested that PFDA might alter metabolic processes at the cellular level and result in the severe body wasting characteristic of PFDA toxicity. In order to test this hypothesis, experiments were carried out that examined the effect of PFDA on respiratory activity in isolated rat liver mitochondria. PFDA produced a concentration-dependent increase in state 4 oxygen consumption, released mitochondrial state 3 respiration from inhibition by oligomycin, and stimulated latent ATPase activity. These results indicate an uncoupling of electron transport and oxidative phosphorylation. High concentrations of PFDA produced progressively less increase in state 4 oxygen consumption and prevented the uncoupling effect of 2,4-dinitrophenol, suggesting additional actions of PFDA on energy-transducing functions of isolated mitochondria.
METHODS Male Wistar rats (150-200 g) were sacrificed by decapitation following halothane anesthesia, the livers were removed, and mitochondria were isolated according to the method of Hogeboom (1955). The final pellet was resuspended in the original buffer minus EDTA to a final protein concentration of 10 mg/ml. In vitro mitochondrial respiration rates were determined polarographically in a sealed 2.0 ml water-jacketed chamber maintained at 25°C using a Clark oxygen electrode and a Gilson Oxygraph recorder. The reaction mixture contained 0.2 M sucrose, 20 mM KG, 5 mM potassium phosphate (pH 7.4), 3 mM MgCI2, 5 mJW succinate as substrate, and 600700 jtg mitochondrial protein. State 3 (phosphorylating) respiration was determined by the rate of oxygen consumption following addition of 150 nM ADP, and state 4 (nonphosphorylating) respiration was determined by the rate of oxygen consumption following depletion of added ADP. Respiratory control ratios (state 3/state 4) ranged from 3.43 to 6.36 (mean 4.94 ± 0.43) and P/O ratios ranged from 1.32 to 1.95 (mean 1.58 ± 0.06). The ability of PFDA to release state 3 respiration from inhibition by
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oligomycin was tested in mitochondria in which 1 pg/ml of oligomycin was added prior to addition of 150 fiM ADR Oligomycin-sensitive latent ATPase activity was determined using the coupled enzyme reaction method of Schwartz et al. (1969). Mitochondria were incubated in a spectrophotometer at 37°C under the following conditions: 5 mM MgCI2,100 mM NaCI, 10 mM KCI, 25 mM Tris-HCI (pH 7.4), 2.5 mMTris-ATP, 0.5 mM NADH, 2i5 mM phosphoenolpyruvic acid, and 20 fi\ of a suspension of pyruvate kinase and lactate dehydrogenase (Sigma) with or without 1.0 /*g/ml oligomycin. The reaction was started with the addition of 300 fig mitochondrial protein. RESULTS In isolated rat liver mitochondria PFDA produced a concentrationdependent increase in state 4 respiration (Fig. 1). There was a linear increase in oxygen consumption up to a PFDA concentration of 87.5 ^g/ml. However, the increase in state 4 oxygen consumption became progressively less at higher concentrations of PFDA (Fig. 2). These data, which suggest an uncoupling of electron transport and oxidative phosphorylation, were supported by the observation that PFDA (100 ^g/ml) was able
FIGURE 1. Polarographic tracings showing the stimulation of state 4 respiration in isolated rat liver mitochondria by different concentrations of PFDA: (1) 12.5, (2) 25, (3) 37.5, (4) 50, (5) 75, and (6) 87.5 /ig/ml. The dotted line indicates the effects of 50 fiM 2,4-dinitrophenol (DNP). MIT, mitochondria (650 ng protein); SUCC, succinate (5 ftM); ADP, 150 nM.
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i
60 80 100 PFDA Oug/ml)
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FIGURE 2. Effect of PFDA on the rate of oxygen consumption during state 4 respiration in isolated rat liver mitochondria. The graph shows the increase above basal state 4 respiration produced by addition of PFDA.
to release mitochondria from inhibition of state 3 respiration produced by exposure to oligomycin (Fig. 3). Oligomycin-sensitive ATPase activity of mitochondria is normally very low but increases dramatically when electron transport and oxidative phosphorylation are uncoupled (Lehninger, 1978). PFDA produced a concentration-dependent stimulation of mitochondrial latent ATPase activity, which was completely inhibited by oligomycin up to 70 /*g/ml PFDA (Fig. 4). A portion of the ATPase activity produced by concentrations of PFDA greater than 70 jig/ml was insensitive to oligomycin. The observation that concentrations of PFDA greater than 87.5 /xg/ml produced progressively less stimulation of state 4 oxygen consumption suggested additional actions of PFDA on mitochondrial respiration. Figure 5 shows that a high concentration of PFDA (150 jig/ml) produced little change in state 4 oxygen consumption but completely inhibited ADPstimulated, state 3 respiration and prevented the uncoupling action of 50 IxM 2,4-dinitrophenol (DNP). These results suggest an inhibition of electron transport at higher PFDA concentrations. DISCUSSION AND CONCLUSIONS The results of these experiments demonstrate that PFDA has the ability to disrupt metabolic processes in isolated rat liver mitochondria. Increases in state 4 (nonphosphorylating) oxygen consumption, release of
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FIGURE 3. Release of oligomycin-inhibited state 3 respiration by PFDA (100 ng/ml). Oligomycin concentration 1 ^g/ml. See Fig. 1 for details.
3.50
10
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30
40
50
60
80
100
[PFDA] /jg/ml FIGURE 4. Stimulation of latent ATPase activity in rat liver mitochondria by PFDA. Activity was determined in the absence (•, n - 5) or presence (O, n - 5) of oligomycin (1ftg/ml). The graph shows the increase above basal ATPase activity produced by addition of PFDA.
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(15(Hig/mD ADP
FIGURE 5. Effect of PFDA (150 /ig/m\) on state respiration and inhibition of the uncoupling action of 2,4-dinitrophenol (50 nM): MIT, mitochondria (638 ng protein). See Fig. 1 for details.
oligomycin-inhibited state 3 respiration, and stimulation of oligomycinsensitive latent ATPase activity indicate that PFDA can act as an uncoupler of electron transport and oxidative phosphorylation. Although the mechanism of this effect cannot be determined from the present data, PFDA does possess an ionizable acidic group similar to uncouplers like 2,4-dinitrophenol and may act as a protonophoric uncoupler to dissipate the high-energy state generated by electron transport (Mitchell, 1966). The observations that the higher end of the PFDA concentration range caused progressively less stimulation of state 4 oxygen consumption and stimulated ATPase activity that was not completely inhibited by oligomycin indicate that PFDA may produce effects in addition to uncoupling electron transport and oxidative phosphorylation. Although a nonspecific disruption of mitochondrial membranes cannot be ruled out, the observation that a single, high concentration of PFDA completely inhibited ADP-stimulated oxygen consumption (state 3 respiration) and prevented the uncoupling action of 2,4-DNP suggests that PFDA may also inhibit electron transport (Lehninger, 1978). The combination of uncoupling of oxidative phosphorylation concomitant with inhibition of electron transport would severely disrupt normal energy production and could result in the "wasting syndrome" characteristic of PFDA toxicity. These effects may be due to the reported ability of PFDA to alter the structural integrity of membranes, which could lead to a disruption of the functional activities observed in isolated mitochondria. Several studies have shown that PFDA can affect in vivo lipid metabolism, including shifts in fatty acid composition of liver (Olson and Andersen, 1983) and
Downloaded by [University of Florida] at 12:31 06 November 2015
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heart membrane phospholipids (Pilcher et al., 1987), as well as tissue cholesterol levels (George and Andersen, 1986) and induction of hepatic peroxisomal fatty acid oxidizing capacity (van Rafelghem et al., 1985; Harrison et al., 1988) parallel with an increase in the capacity for fatty acid biosynthesis (Kelling et al., 1987). Interactions with lipids may be responsible for the changes in functional membrane-mediated activites observed in animals treated with PFDA. These effects include decreased numbers of /3-receptor binding sites, reduced responsiveness of adenylate cyclase to norepinephrine stimulation, and altered thermodynamics of adenylate cyclase in rat heart membranes (Pilcher et al., 1987). Several reports have demonstrated alterations in membrane-mediated activities following acute in vitro exposures to PFDA. These include inactivation of a membrane transport channel in L 5178 Y mouse lymphoma cells (Wigler and Shah, 1986); altered surface membrane expression and immunoglobulin secretion in human and murine /3-cell lymphomas (Levitt and Liss, 1986); inhibition of binding of the fluorescent dye merocyanine 540 to plasma membrane phospholipids from human j8-lymphoblast cells (Levitt and Liss, 1987); and decreased osmotic fragility and increased fluidity of red blood cell membranes (Olson et al., 1983). These in vitro effects are thought to be due to interactions of PFDA with membrane lipids. The effects on mitochondrial energy-transducing functions observed in the present study may likewise result from an interaction of PFDA with lipid components of mitochondrial membranes that are critical to normal metabolic processes. REFERENCES Bryce, H. G. 1964. Industrial and utilitarian aspects of fluorine chemistry. In Fluorine Chemistry, vol. V, ed. J. H. Simons, pp. 297-498. New York: Academic Press. George, M. E., and Andersen, M. E. 1986. Toxic effects of perfluoro-n-decanoic acid (PFDA) in rats. Toxicol. Appl. Pharmacol. 85:169-180. Gutshall, D. M., Pilcher, G. D., and Langley, A. E. 1988. Effect of thyroxine supplementation on the response to perfluorodecanoic acid (PFDA) in rats. J. Toxicol. Environ. Health 24:491-498. Harrison, E. H., Lane, J. S., van Rafelghem, M. J., and Andersen, M. E. 1988. Perfluoro-n-decanoic acid: Induction of peroxisomal β-oxidation by a fatty acid with dioxin-like toxicity. Lipids 23:115-119. Hogeboom, G. H. 1955. Fractionation of cell components of animal tissues. Methods Enzymol. 1:16-19. Kelling, C. K., van Rafelghem, M. J., Menahan, L. A., and Peterson, R. E. 1987. Effects of perfluoradecanoic acid on hepatic indices of thyroid status. Biochem. Pharmacol. 36:487-496. Langley, A. E., and Pilcher, G. D. 1985. Thyroid, cardiac, and hypothermic effects of perfluoro-ndecanoic acid (PFDA) in rats. J. Toxicol. Environ. Health 15:485-491. Lehninger, A. L. 1978. Oxidative phosphorylation, mitochondrial structure and the compartmentation of respiratory metabolism. In Biochemistry, pp. 520-521, 531-534, 544, 667, 840-845, and 884. New York: Worth. Levitt, D., and Liss, A. 1986. Toxicity of perfluorinated fatty acids for human and murine β cell lines. Toxicol. Appl. Pharmacol. 96:1-11.
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Levitt, D., and Liss, A. 1987. Perfluorinated fatty acids alter merocyanine 540 dye binding to plasma membranes. J. Toxicol. Environ. Health 20:303-316. Mitchell, P. 1966. Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, pp. 135-156. Bodmin, U.K.: Clynn Research Ltd. Olson, C. T., and Andersen, M. E. 1983. The acute toxicity of perfluoro-octanoic and perfluorodecanoic acids in male rats and effects on tissue fatty acids. Toxicol. Appl. Pharmacol. 70:362-372. Olson, C. T., George, M. E., and Andersen, M. E. 1983. Effect of perfluoro-n-decanoic acid on cell composition and membranes. Toxicologist 3:99. Pilcher, C. D., Gutshall, D. M., and Langley, A. E. 1987. Effects of PFDA on rat heart β-receptors, adenylate cylase, and fatty acid composition. Toxicol. Appl. Pharmacol. 90:198-205. Schwartz, A., Allen, J. C. Harigaya, S. 1969. Possible involvement of cardiac Na + – K+ – ATPase in the mechanism of action of cardiac glycosides. J. Pharmacol. Exp. Then 168:31-41. Shinoda, K., and Normura, T. 1980. Miscibillty of fluorocarbon and hydrocarbon surfactants in micelles and liquid mixtures. Basic studies of oil repellent and fire extinguishing agents. J. Phys. Chem. 84:365-369. Van Rafelghem, M. J., Vanden Heuvel, J. P., Menahan, L. A., and Peterson, R. E. 1988. Perfluorodecanoic acid and lipid metabolism in the rat lipids. 23:671-678. Van Rafelghem, M. J., Noren, C. W., Menahan, L. A., and Peterson, R. E. 1988. Interrelationships between energy and fat metabolism and hypophagia in rats treated with perfluorodecanoic acid. Toxicol. Lett. 40:57-69. Wigler, P. W., and Shah, Y. B. 1986. Perfluorodecanoic acid inactivation of a channel for 2aminopurine in the L 5178 Y cell membrane and recovery of the channel. Toxicol. Appl. Pharamcol. 85:456-463. Received June 7, 1989 Accepted October 29, 1989
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effects of perfluoro‐n‐decanoic acid on the respiratory activity of isolated rat liver mitochondria Albert E. Langley To cite this article: Albert E. Langley (1990) Effects of perfluoro‐n‐decanoic acid on the respiratory activity of isolated rat liver mitochondria, Journal of Toxicology and Environmental Health, 29:3, 329-336, DOI: 10.1080/15287399009531395 To link to this article: http://dx.doi.org/10.1080/15287399009531395
Published online: 20 Oct 2009.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 16 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 06 November 2015, At: 12:31
EFFECTS OF PERFLUORO-n-DECANOIC ACID ON THE RESPIRATORY ACTIVITY OF ISOLATED RAT LIVER MITOCHONDRIA Albert E. Langley
Downloaded by [University of Florida] at 12:31 06 November 2015
Department of Pharmacology and Toxicology, Wright State University, School of Medicine, Dayton, Ohio
The toxic responses of rats to a single dose of perfluorodecanoic acid (PFDA) include reduced food consumption, severe body weight loss ("wasting syndrome"), and hypothermia. Recent studies have suggested that some of these effects may be due to alterations of basic metabolic processes in animals treated with PFDA. In order to test this hypothesis the effects of PFDA on respiratory activity of isolated rat liver mitochondria were examined. PFDA concentrations up to 87.5 µg/ml produced a linear increase in oxygen consumption during state 4 (nonphosphorylating) respiration. This suggested uncoupling of electron transport and oxidative phosphorylation was supported by the observation that PFDA released mitochondrial state 3 respiration from inhibition by oligomycin and stimulated latent ATPase activity. Concentrations of PFDA greater than 87.5 µg/ml produced progressively less increase in state 4 oxygen consumption, and a single high concentrations (150 µg/ml) completely inhibited state 3 respiration and prevented the uncoupling effect of 2,4-dinitrophenol. These observations suggest that in addition to uncoupling electron transport and oxidative phosphorylation PFDA may affect other energy-transducing functions of liver mitochondria such as inhibiting electron transport. These effects on mitochondrial respiration may help to explain the "wasting syndrome" characteristic of PFDA toxicity.
INTRODUCTION Perfluoro-n-decanoic acid (PFDA) is a member of a family of perfluorinated carboxylic acids that have found widespread commercial applications as lubricants, surfactants, wetting agents, corrosion inhibitors, and gloss enhancers of commercial aqueous emulsion type floor waxes (Bryce, 1964). Perfluorinated fatty acid surfactants have also been used to impart oil and water repellancy to cloth, leather, and paper. They are found in aqueous film-forming foam fire extinguishants (Shinoda and Nomura, 1980). In rats a single dose of PFDA near the LD50 has been reported to produce hypophagia and severe weight loss (Olson and Andersen, 1983; This work was supported by a grant from the Air Force Office of Scientific Research, AFOSR82-0264.
329 Journal of Toxicology and Environmental Health, 29:329-336, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
Downloaded by [University of Florida] at 12:31 06 November 2015
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Langley and Pilcher, 1985), bradycardia, hypothermia, and decreased serum thyroid hormone levels (Langley and Pilcher, 1985). Serum thyroxine levels were significantly lower than control levels as early as 12 h after a single 75 mg/kg dose of PFDA, and were maximally reduced within 48 h (Langley and Pilcher, 1985). These data indicated that decreases in serum thyroid hormone levels were one of the earliest responses to a single injection of PFDA. Consequently rats were supplemented with thyroxine in an effort to prevent the toxic effects of PFDA. Supplementing with 200 ^g/kg-d thyroxine prevented PFDA-induced hypophagia but was ineffective at preventing the "wasting syndrome" and hypothermia (Gutshall et al., 1988). These observations coupled with the reports that PFDA-treated rats lost significantly greater weight than pair-fed controls (Langley and Pilcher, 1985; Olsen and Andersen, 1983; Van Rafelghem et al., 1988) suggested that PFDA might alter metabolic processes at the cellular level and result in the severe body wasting characteristic of PFDA toxicity. In order to test this hypothesis, experiments were carried out that examined the effect of PFDA on respiratory activity in isolated rat liver mitochondria. PFDA produced a concentration-dependent increase in state 4 oxygen consumption, released mitochondrial state 3 respiration from inhibition by oligomycin, and stimulated latent ATPase activity. These results indicate an uncoupling of electron transport and oxidative phosphorylation. High concentrations of PFDA produced progressively less increase in state 4 oxygen consumption and prevented the uncoupling effect of 2,4-dinitrophenol, suggesting additional actions of PFDA on energy-transducing functions of isolated mitochondria.
METHODS Male Wistar rats (150-200 g) were sacrificed by decapitation following halothane anesthesia, the livers were removed, and mitochondria were isolated according to the method of Hogeboom (1955). The final pellet was resuspended in the original buffer minus EDTA to a final protein concentration of 10 mg/ml. In vitro mitochondrial respiration rates were determined polarographically in a sealed 2.0 ml water-jacketed chamber maintained at 25°C using a Clark oxygen electrode and a Gilson Oxygraph recorder. The reaction mixture contained 0.2 M sucrose, 20 mM KG, 5 mM potassium phosphate (pH 7.4), 3 mM MgCI2, 5 mJW succinate as substrate, and 600700 jtg mitochondrial protein. State 3 (phosphorylating) respiration was determined by the rate of oxygen consumption following addition of 150 nM ADP, and state 4 (nonphosphorylating) respiration was determined by the rate of oxygen consumption following depletion of added ADP. Respiratory control ratios (state 3/state 4) ranged from 3.43 to 6.36 (mean 4.94 ± 0.43) and P/O ratios ranged from 1.32 to 1.95 (mean 1.58 ± 0.06). The ability of PFDA to release state 3 respiration from inhibition by
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Downloaded by [University of Florida] at 12:31 06 November 2015
oligomycin was tested in mitochondria in which 1 pg/ml of oligomycin was added prior to addition of 150 fiM ADR Oligomycin-sensitive latent ATPase activity was determined using the coupled enzyme reaction method of Schwartz et al. (1969). Mitochondria were incubated in a spectrophotometer at 37°C under the following conditions: 5 mM MgCI2,100 mM NaCI, 10 mM KCI, 25 mM Tris-HCI (pH 7.4), 2.5 mMTris-ATP, 0.5 mM NADH, 2i5 mM phosphoenolpyruvic acid, and 20 fi\ of a suspension of pyruvate kinase and lactate dehydrogenase (Sigma) with or without 1.0 /*g/ml oligomycin. The reaction was started with the addition of 300 fig mitochondrial protein. RESULTS In isolated rat liver mitochondria PFDA produced a concentrationdependent increase in state 4 respiration (Fig. 1). There was a linear increase in oxygen consumption up to a PFDA concentration of 87.5 ^g/ml. However, the increase in state 4 oxygen consumption became progressively less at higher concentrations of PFDA (Fig. 2). These data, which suggest an uncoupling of electron transport and oxidative phosphorylation, were supported by the observation that PFDA (100 ^g/ml) was able
FIGURE 1. Polarographic tracings showing the stimulation of state 4 respiration in isolated rat liver mitochondria by different concentrations of PFDA: (1) 12.5, (2) 25, (3) 37.5, (4) 50, (5) 75, and (6) 87.5 /ig/ml. The dotted line indicates the effects of 50 fiM 2,4-dinitrophenol (DNP). MIT, mitochondria (650 ng protein); SUCC, succinate (5 ftM); ADP, 150 nM.
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i
60 80 100 PFDA Oug/ml)
i
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FIGURE 2. Effect of PFDA on the rate of oxygen consumption during state 4 respiration in isolated rat liver mitochondria. The graph shows the increase above basal state 4 respiration produced by addition of PFDA.
to release mitochondria from inhibition of state 3 respiration produced by exposure to oligomycin (Fig. 3). Oligomycin-sensitive ATPase activity of mitochondria is normally very low but increases dramatically when electron transport and oxidative phosphorylation are uncoupled (Lehninger, 1978). PFDA produced a concentration-dependent stimulation of mitochondrial latent ATPase activity, which was completely inhibited by oligomycin up to 70 /*g/ml PFDA (Fig. 4). A portion of the ATPase activity produced by concentrations of PFDA greater than 70 jig/ml was insensitive to oligomycin. The observation that concentrations of PFDA greater than 87.5 /xg/ml produced progressively less stimulation of state 4 oxygen consumption suggested additional actions of PFDA on mitochondrial respiration. Figure 5 shows that a high concentration of PFDA (150 jig/ml) produced little change in state 4 oxygen consumption but completely inhibited ADPstimulated, state 3 respiration and prevented the uncoupling action of 50 IxM 2,4-dinitrophenol (DNP). These results suggest an inhibition of electron transport at higher PFDA concentrations. DISCUSSION AND CONCLUSIONS The results of these experiments demonstrate that PFDA has the ability to disrupt metabolic processes in isolated rat liver mitochondria. Increases in state 4 (nonphosphorylating) oxygen consumption, release of
Downloaded by [University of Florida] at 12:31 06 November 2015
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FIGURE 3. Release of oligomycin-inhibited state 3 respiration by PFDA (100 ng/ml). Oligomycin concentration 1 ^g/ml. See Fig. 1 for details.
3.50
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40
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100
[PFDA] /jg/ml FIGURE 4. Stimulation of latent ATPase activity in rat liver mitochondria by PFDA. Activity was determined in the absence (•, n - 5) or presence (O, n - 5) of oligomycin (1ftg/ml). The graph shows the increase above basal ATPase activity produced by addition of PFDA.
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(15(Hig/mD ADP
FIGURE 5. Effect of PFDA (150 /ig/m\) on state respiration and inhibition of the uncoupling action of 2,4-dinitrophenol (50 nM): MIT, mitochondria (638 ng protein). See Fig. 1 for details.
oligomycin-inhibited state 3 respiration, and stimulation of oligomycinsensitive latent ATPase activity indicate that PFDA can act as an uncoupler of electron transport and oxidative phosphorylation. Although the mechanism of this effect cannot be determined from the present data, PFDA does possess an ionizable acidic group similar to uncouplers like 2,4-dinitrophenol and may act as a protonophoric uncoupler to dissipate the high-energy state generated by electron transport (Mitchell, 1966). The observations that the higher end of the PFDA concentration range caused progressively less stimulation of state 4 oxygen consumption and stimulated ATPase activity that was not completely inhibited by oligomycin indicate that PFDA may produce effects in addition to uncoupling electron transport and oxidative phosphorylation. Although a nonspecific disruption of mitochondrial membranes cannot be ruled out, the observation that a single, high concentration of PFDA completely inhibited ADP-stimulated oxygen consumption (state 3 respiration) and prevented the uncoupling action of 2,4-DNP suggests that PFDA may also inhibit electron transport (Lehninger, 1978). The combination of uncoupling of oxidative phosphorylation concomitant with inhibition of electron transport would severely disrupt normal energy production and could result in the "wasting syndrome" characteristic of PFDA toxicity. These effects may be due to the reported ability of PFDA to alter the structural integrity of membranes, which could lead to a disruption of the functional activities observed in isolated mitochondria. Several studies have shown that PFDA can affect in vivo lipid metabolism, including shifts in fatty acid composition of liver (Olson and Andersen, 1983) and
Downloaded by [University of Florida] at 12:31 06 November 2015
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heart membrane phospholipids (Pilcher et al., 1987), as well as tissue cholesterol levels (George and Andersen, 1986) and induction of hepatic peroxisomal fatty acid oxidizing capacity (van Rafelghem et al., 1985; Harrison et al., 1988) parallel with an increase in the capacity for fatty acid biosynthesis (Kelling et al., 1987). Interactions with lipids may be responsible for the changes in functional membrane-mediated activites observed in animals treated with PFDA. These effects include decreased numbers of /3-receptor binding sites, reduced responsiveness of adenylate cyclase to norepinephrine stimulation, and altered thermodynamics of adenylate cyclase in rat heart membranes (Pilcher et al., 1987). Several reports have demonstrated alterations in membrane-mediated activities following acute in vitro exposures to PFDA. These include inactivation of a membrane transport channel in L 5178 Y mouse lymphoma cells (Wigler and Shah, 1986); altered surface membrane expression and immunoglobulin secretion in human and murine /3-cell lymphomas (Levitt and Liss, 1986); inhibition of binding of the fluorescent dye merocyanine 540 to plasma membrane phospholipids from human j8-lymphoblast cells (Levitt and Liss, 1987); and decreased osmotic fragility and increased fluidity of red blood cell membranes (Olson et al., 1983). These in vitro effects are thought to be due to interactions of PFDA with membrane lipids. The effects on mitochondrial energy-transducing functions observed in the present study may likewise result from an interaction of PFDA with lipid components of mitochondrial membranes that are critical to normal metabolic processes. REFERENCES Bryce, H. G. 1964. Industrial and utilitarian aspects of fluorine chemistry. In Fluorine Chemistry, vol. V, ed. J. H. Simons, pp. 297-498. New York: Academic Press. George, M. E., and Andersen, M. E. 1986. Toxic effects of perfluoro-n-decanoic acid (PFDA) in rats. Toxicol. Appl. Pharmacol. 85:169-180. Gutshall, D. M., Pilcher, G. D., and Langley, A. E. 1988. Effect of thyroxine supplementation on the response to perfluorodecanoic acid (PFDA) in rats. J. Toxicol. Environ. Health 24:491-498. Harrison, E. H., Lane, J. S., van Rafelghem, M. J., and Andersen, M. E. 1988. Perfluoro-n-decanoic acid: Induction of peroxisomal β-oxidation by a fatty acid with dioxin-like toxicity. Lipids 23:115-119. Hogeboom, G. H. 1955. Fractionation of cell components of animal tissues. Methods Enzymol. 1:16-19. Kelling, C. K., van Rafelghem, M. J., Menahan, L. A., and Peterson, R. E. 1987. Effects of perfluoradecanoic acid on hepatic indices of thyroid status. Biochem. Pharmacol. 36:487-496. Langley, A. E., and Pilcher, G. D. 1985. Thyroid, cardiac, and hypothermic effects of perfluoro-ndecanoic acid (PFDA) in rats. J. Toxicol. Environ. Health 15:485-491. Lehninger, A. L. 1978. Oxidative phosphorylation, mitochondrial structure and the compartmentation of respiratory metabolism. In Biochemistry, pp. 520-521, 531-534, 544, 667, 840-845, and 884. New York: Worth. Levitt, D., and Liss, A. 1986. Toxicity of perfluorinated fatty acids for human and murine β cell lines. Toxicol. Appl. Pharmacol. 96:1-11.
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Levitt, D., and Liss, A. 1987. Perfluorinated fatty acids alter merocyanine 540 dye binding to plasma membranes. J. Toxicol. Environ. Health 20:303-316. Mitchell, P. 1966. Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, pp. 135-156. Bodmin, U.K.: Clynn Research Ltd. Olson, C. T., and Andersen, M. E. 1983. The acute toxicity of perfluoro-octanoic and perfluorodecanoic acids in male rats and effects on tissue fatty acids. Toxicol. Appl. Pharmacol. 70:362-372. Olson, C. T., George, M. E., and Andersen, M. E. 1983. Effect of perfluoro-n-decanoic acid on cell composition and membranes. Toxicologist 3:99. Pilcher, C. D., Gutshall, D. M., and Langley, A. E. 1987. Effects of PFDA on rat heart β-receptors, adenylate cylase, and fatty acid composition. Toxicol. Appl. Pharmacol. 90:198-205. Schwartz, A., Allen, J. C. Harigaya, S. 1969. Possible involvement of cardiac Na + – K+ – ATPase in the mechanism of action of cardiac glycosides. J. Pharmacol. Exp. Then 168:31-41. Shinoda, K., and Normura, T. 1980. Miscibillty of fluorocarbon and hydrocarbon surfactants in micelles and liquid mixtures. Basic studies of oil repellent and fire extinguishing agents. J. Phys. Chem. 84:365-369. Van Rafelghem, M. J., Vanden Heuvel, J. P., Menahan, L. A., and Peterson, R. E. 1988. Perfluorodecanoic acid and lipid metabolism in the rat lipids. 23:671-678. Van Rafelghem, M. J., Noren, C. W., Menahan, L. A., and Peterson, R. E. 1988. Interrelationships between energy and fat metabolism and hypophagia in rats treated with perfluorodecanoic acid. Toxicol. Lett. 40:57-69. Wigler, P. W., and Shah, Y. B. 1986. Perfluorodecanoic acid inactivation of a channel for 2aminopurine in the L 5178 Y cell membrane and recovery of the channel. Toxicol. Appl. Pharamcol. 85:456-463. Received June 7, 1989 Accepted October 29, 1989
