Covalent binding of perfluorinated fatty acids to proteins in the plasma, liver and testes of rats

Chem.-Biol. Interactions, 82 (1992) 317-328 Elsevier ScientificPublishers Ireland Ltd.

317

COVALENT BINDING OF PERFLUORINATED FATTY ACIDS TO PROTEINS IN THE PLASMA, LIVER AND TESTES OF RATS JOHN P. VANDEN HEUVELa~*, BENEDICT I. KUSLIKISb,** and RICHARDE. PETERSONa'b aEnvironmental To'zicologyCenter and bSchoolof Pharmacy, University of Wisconsin, Madison, WI 58706 (USA)

(Received October 18th, 1991) (Revision received February 26th, 1992) (Accepted February 28th, 1992)

SUMMARY Perfluorinated fatty acids alter hepatic lipid metabolism and are potent peroxisome proliferators in rodents. Two such perfluorinated acids, perfluorodecanoic acid (PFDA) and perfluorooctanoic acid (PFOA), were examined to determine if they covalently bind cellular proteins. PFDA and PFOA were found to covalently bind proteins when administered to rats in vivo. The liver, plasma and testes of male rats treated with [1-14C]PFDA or PFOA (9.4 ~mol/kg) contained detectable levels of covalently bound 14C (0.1-0.5% of the tissue 14C content). Characterization of PFDA covalent binding to albumin in vitro showed that cysteine significantly decreased binding with no effect of methionine, suggesting protein sulfhydryl groups are involved. In cytosolic and microsomal incubation there was no effect of the addition of CoA, ATP or NADPH on the magnitude of the covalent binding of PFDA. Therefore PFDA need not be metabolically activated to form covalent adducts. Despite demonstration of covalent binding of PFDA and PFOA to proteins both in vivo and in vitro, the role of this macromolecular binding in perfluorinated fatty acid toxicity is not known.

Key wards: Perfluorodecanoic acid -- Perfluorooctanoic acid -- Covalent binding -- Peroxisome proliferation -- Liver -- Testis -- Plasma C o v ' r e s p ~ e to: R.E. Peterson, School of Pharmacy, 425 N. Charter St., Madison,WI 53706, USA. *Present address: NIEHS, P.O. Box 12233 MD-D404, Research Triangle Park, NC 27709, USA. **Present address: Blodgett Regional Poison Center, Blodgett Memorial Medical Center, 1840 Wealthy S.E., Grand Rapids, MI 49506, USA.

0009-2797/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland

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INTRODUCTION

Perfluorinated fatty acids, such as perfluorodecanoic acid (PFDA) and perfluorooctanoic acid (PFOA), are used industrially due to their surface active properties. PFDA and PFOA alter hepatic lipid metabolism [1,2] and are potent peroxisome proliferators [2,3] in rodents. Perfluorinated fatty acids are considered to be metabolically inert with neither PFDA nor PFOA being metabolized to lipid conjugates or to water soluble metabolites in male or female rats [4,5]. Nonetheless, following in vivo administration, PFDA's partitioning behavior in organic solvents fluctuates depending on the time since exposure, suggesting that PFDA exists in at least two forms in the liver [6]. One possible explanation for this change in partitioning is that PFDA is covalently bound to hepatocellular proteins. Both PFDA and PFOA are persistent in male rats [4,5] and are highly protein bound [7,8], although the amount of covalent binding of these compounds is not known. The primary objective of this study is to determine if PFDA or PFOA covalently bind to proteins following in vivo administration. Also, the nature of this association will be examined in vitro to determine the various factors involved in perfluorinated fatty acid-protein adduct formation. We will demonstrate that PFDA binds covalently to proteins in vivo and in primary rat hepatocytes in culture, although the low specific activity of the [1-14C]perfluorinated fatty acid precludes identification of specifically modified proteins. Based on in vitro data, this protein-PFDA association appears to be non-enzymatic, i.e does not require CoA, ATP or NADPH and may involve protein sulfhydryl groups. MATERIALSAND METHODS Reagents and chemicals [1-14C]Perfluorodecanoic acid (99% pure, spec. act. 55 mCi/mmol) and [1-14C]perfluorooctanoic acid (99% pure, spec. act. 51.7 mCi/mmol) were synthesized and purified as described previously [9]. Hemoglobin (2 x crystallized, dialyzed and lyophilized), fatty acid free bovine serum albumin and all tissue culture media and supplements were from Sigma Chemical Co. (St. Louis, MO). Reagent grade solvents were obtained from Aldrich Chemical Co. (Milwaukee, WI). Water was purified by passing distilled water through a 4-bowl Milli-Q water purification system and 0.25-#m filter (Millipore Corporation, Milford,

MA). Animals and treatment Six-week old male Harlan Sprague- Dawley rats (170-195 g), obtained from Harlan Sprague-Dawley (Indianapolis, IN), were individually housed in suspended stainless-steel cages in a temperature-controlled room (-21°C) with a 12-h light/dark cycle (lighted, 05:00 h - 17:00 h). Feed (Purina Rat Chow, No. 5012, Ralston Purina Co., St. Louis, MO) and water were provided ad libitum throughout the study. An acclimation period of at least 1 week was allowed before the initiation of an experiment.

319

Covalent binding of PFDA and PFOA to proteins in vivo [1-i4C]PFDA or PFOA was administered to rats in propylene glycollwater (1:1, v/v; 1 ml/kg) at a dose of 9.4 tLmollkg, i.p. At designated times posttreatment, rats were anesthetized with Nembutal (50 mg/kg, i.p.), tissues were quickly removed, freeze clamped [10] and stored at -70°C. Blood was removed in a lightly heparinized needle and plasma separated by centrifugation. Macromolecular binding was determined on tissue homogenates which were brought up to 10% TCA and put on ice for 10 rain. To remove any unbound perfluorinated acid, samples were washed with 3 ml methanol/ether (3:1) 6 times and then 3 more times using 3 ml of ethyl acetate. Washing was continued ff necessary until no additional PFDA- or PFOA-derived i4C could be extracted from the protein. One milliliter of I N NaOH was added to the protein precipitate and placed in a shaking water bath overnight at 37°C to solubilize it. The sample was then neutralized with 1 M acetic acid and i ml was used for liquid scintillation counting while another aliquot was used for protein analysis [11]. PFDA- or PFOA-derived radioactivity in all samples was quantitated using a Packard Liquid Scintillation Analyzer (Model 2000CA) with quench correction performed with a Packard DPM 1-2-3 software program. Radioactivity in hydrolysates was determined in Hionic-Fluor scintillation cocktail. Binding was expressed as pmol PFDA or PFOA equiv./mg protein. Covalent binding of PFDA and PFOA to hemoglobin and albumin in vitro To screw top tubes containing albumin or hemoglobin in 1.0 ml of phosphate buffer, either 14C-labeled PFDA or PFOA were added to give a 2, 4, 8 or 100 ~M solution. If methionine or cysteine were present, they were added in I ml of phosphate buffer to bring the reaction mixture up to 2 ml. All experiments were done using a final volume of buffer of 2 ml. The time of incubation was 1 h at 37°C. Protein was precipitated with 10% TCA. To remove any free unbound perfluorinated acid, samples were washed with 5 ml of acetone 5 times. Samples were vortexed for 1 rain and then spun at 2500 rev./min for 5 rain after each acetone wash step. The acetone was transferred off. Protein samples were then washed with 5 ml of ether 2 additional times. Washing was continued ff necessary until no additional PFDA or PFOA (as determined by liquid scintillation counting) could be extracted from the protein. The protein powder was hydrolyzed with 1 ml of I N NaOH at 37°C in a shaking water bath overnight. Binding was expressed as pmol PFDA or PFOA equiv./rag of protein. Covalent binding of PFDA to rat liver cytosolic or microsome preparations in vitro Microsomal and cytosolic fractions were prepared from the liver of male rats. The livers were minced and homogenized in a Potter-Elvehjem homogenizer in KCI solution to yield a homogenate of 250 mg liver wet weight/ml. Cytosol was obtained by differential centrifugation of the homogenate through a 9000 × g supernatant and then precipitation at 105 000 x g. The 105 000 x g pellet was thoroughly washed by mixing with 0.1 M sodium pyrophosphate containing 0.3 M sucrose (pH 7.5), followed by a second centrifugation at 105 000 x g. The

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washed microsomes were suspended in 0.05 M Tris-HC1 buffer (pH 7.4), to give a protein concentration of 10 mg/ml. For routine use, this preparation was dispensed into 1-ml capped vials. After purging with Ar, the vials were stored at - 7 0 ° C . Covalent binding to cytosolic and microsomal preparations was determined in a final reaction mixture (0.2 ml) in 150 mM Tris-HC1 containing combinations of 2.5 mM ATP, 0.6 mM CoA or i mM NADPH with [1-14C]PFDA (0.1 mM) and the protein sample (50 ~g). The reaction proceeded for 30 min at 37°C and was terminated by adding 1 ml ice-cold acetone. Protein samples were washed 3-times with acetone, three times with chloroform/methanol (2:1, v/v) and twice with ethanol. The final ethanol wash did not remove any detectable levels of radioactivity. Values are expressed as pmol PFDA or PFOA equiv./mg protein.

Statistical analysis Differences from control were determined by one-way analysis of variance. The least significant differences test was used to compare individual means where analysis of variance indicated differences. The level of significance for all analyses was P < 0.05. RE SULTS

Covalent binding of PFDA and PFOA to target tissues in rats The extent of PFDA and PFOA covalent binding to liver, plasma and testis proteins of rats is shown in Fig. 1. These tissues were selected because they are either major tissues of distribution for PFDA or PFOA in rats (liver and plasma), or they are target organs for perfluorinated acid toxicity (liver and testes). The covalent binding data for each perfluorinated acid for each tissue (2 h, 1 and 4 days after treatment) were pooled because no time-dependent changes in the absolute and relative concentrations of covalently bound protein were observed (data not shown). Also, the tissue elimination half-lives for PFDA and PFOA (average tissue t1/2 25 days for PFDA [4] and 9 days for PFOA [5]) show that there is little difference in tissue concentrations of perfluorinated fatty acids in the range 2 h to 4 days. The absolute concentration of covalently bound protein in the liver was significantly higher in PFDA- than PFOA-treated rats. The absolute concentration of covalently bound perfluorinated acid in the plasma and testes was similar for both PFDA- and PFOA-treated rats. The liver contained a higher concentration of covalently bound PFDA than did either the plasma or testes. In PFOA-treated rats, the absolute concentration of covalently bound PFOA was significantly higher in the plasma than in the liver. The amount of perfluorinated acid covalently bound to proteins in tissues is also expressed relative to the total tissue concentration of perfluorinated acid to give percent of the tissue concentration which is covalently bound to proteins (pmol covalently bound/pmol total × 100, bottom panel). There was no difference between PFDA- and PFOA-treated rats in the relative concentration of covalently bound PFDA or PFOA in the liver, despite significant differences in

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0.0 UVER PLASMA TESTES Fig. 1. Covalent binding of PFDA- and PFOA-derived 14C to rat liver, plasma and testes protein following in vivo administration. Results are expressed as the absolute concentration of covalently bound PFDA and PFOA (pmol PFDA or PFOA equiv, covalently bound/rag protein, top panel) or as the relative concentration perfluorinated acid derived-14C per tissue (pmol PFDA or PFOA equiv, covalently bouncYpmol of total PFDA or PFOA equiv, x 100, bottom panel). Rats were administered 9.4 ~mol/kg of either [1-14C]PFDA (open bars) or [1A4C]PFOA (closed bars) i.p. and tissues removed 2 h, i and 4 days post-treatment. Since no time dependent changes in either absolute or relative concentrations of covalently bound PFDA- or PFOA-derived 14C were found the data for all times was combined and expressed as mean ± S.E. (n = 10-11). Bars with different superscripts indicate significant differences between the tissues of PFOA-treated rats (a,b) or PFDA-treated rats (c,d). The asterisk indicates significant differences between PFDA and PFOA covalent binding within the same tissue.

the absolute concentration of covalently bound protein in this organ. This reflects the fact that the liver accumulates twice as much PFDA as PFOA following equimolar administration of perfluorinated acid [4,5]. The relative concentrations of PFDA or PFOA covalently bound to proteins in the plasma were similar to those seen in the liver. Of the tissues examined, the testes had the highest relative concentration of PFDA- or PFOA~terived radioactivity covalently

322

bound to protein. Approximately 0.4% of the testes PFDA or PFOA concentration was covalently bound. To determine which proteins were modified following in vivo administration of PFDA and PFOA, tissue were homogenized and subjected to SDS-PAGE/autoradiography with fluorography. Due to the small amount of covalently bound 14C found within these tissues, no radioactive protein bands were detected when 50 ~g protein was separated on SDS-PAGE and the X-ray film was exposed to the gel for up to 2 months at -70°C.

Covalent binding of PFDA and PFOA to hemoglobin and albumin in vitro The binding of perfluorinated fatty acids to proteins was characterized in vitro using albumin as a protein source (Fig. 2). As shown in the left panel, with [1-14C]PFDA concentration held constant (0.1 mM), the covalent binding increased in a linear fashion as albumin concentration increased. At 80 ~M albumin, 80% of the PFDA present in the incubation was covalently bound after 60 min of incubation. This indicates that PFDA and not a contaminant present in small quantity was covalently associated with albumin. In the center panel, the covalent binding of [1-14C]PFDA to a constant concentration of albumin (8 ~M) increased in a linear fashion with increasing PFDA concentrations. In data not shown, PFOA also covalently bound to this protein with similar amounts of covalently bound protein being formed. In the right panel, PFDA and albumin concentrations are held constant and incubation time was varied. PFDA covaleht binding to albumin in vitro showed a time-dependent increase that plateaued at approximately 1 h with no significant increase thereafter for up to 5 h.

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Fig. 2. Characterization of the covalent binding of PFDA to albumin in vitro. Shown in the left panel, albumin ( 0 - 8 0 ~M) was incubated with 0.1 mM [1-14C]PFDA for 60 min. In the center panel various concentration of [1-z4C]PFDA ( 0 - 8 ~M) were incubated with 8/~M albumin for 60 min. In the right panel, 3/~M PFDA was incubated with 8/~M albumin for various periods of time. The amount of PFDA covalently bound to albumin was determined as described in Methods. Results are mean + S.E. (n = 4 - 5 ) .

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The covalent binding of the perfluorinated acids to albumin and hemoglobin was further examined with cysteine or methionine added to the incubations. As shown in Table I, PFDA covalent binding to albumin could be reduced by the addition of 10 mM cysteine. Methionine added to identical incubations had no effect on PFDA covalent binding to albumin. Similarly, addition of cysteine but not methionine to the incubations significantly decreased the covalent binding of PFDA to hemoglobin. The covalent binding of PFOA to hemoglobin was also diminished by the addition of cysteine with no effect of methionine in parallel incubations (data not shown). Thus, the ability of cysteine to inhibit PFOA and PFDA covalent binding of proteins suggests that protein sulfhydryl groups may be involved.

Covalent binding of PFDA to rat liver cytosolic or microsomal preparations In vitro studies were performed to determine if the covalent binding of PFDA to proteins could be accelerated by the addition of cofactors and]or enzyme sources. As shown in Table II, the covalent association of PFDA with proteins was higher in the microsomal fraction than with the cytosolic fraction. The addition of ATP, CoA or NADPH had no effect on the covalent binding of PFDA to proteins of either liver cytosolic or microsomal origin. When liver cytosol and microsome fractions are combined, in amounts identical to those examined in separate incubations, the amount of covalent binding was significantly lower than when microsome preparations were incubated alone. Therefore, the addition of liver cytosol appears to decrease the amount of covalent binding of PFDA to liver microsomal proteins. This cytosolic factor does not appear to be reduced glutathione, as 10 mM GSH had no effect on PFDA covalent binding to microsomes (data not shown).

TABLE I EFFECTS OF METHIONINE OR CYSTEINE ON THE COVALENT BINDING OF PERFLUORODECANOICACIDTO ALBUMINOR HEMOGLOBININ VITRO Protein samples(4 ~M)were incubatedwith 8/~M[I-14C]PFDAwith or withoutI0 mM aminoacids for 1 h at 37°C as described in Materials and Methods. Results are mean ± S.E. (n = 5).

Protein

Amino acid

Covalentlybound PFDA (pmol/mgprotein)

Albumin

None Methionine Cysteine

161 ± 33 220 ± 75 57 ± 11"

Hemoglobin

None Methionine Cysteine

90 ± 32 86 ± 2 22 ± 9*

*Significantlydifferent from samples with no amino acid added, P < 0.05.

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TABLE II EFFECTS OF CoA, ATP AND NADPH ON COVALENT BINDING OF PERFLUORODECANOIC ACID TO RAT LIVER CYTOSOLIC AND/OR MICROSOMAL PREPARATIONS IN VITRO C y t o s o l i c o r m i c r o s o m a l p r e p a r a t i o n s (20 ~ g p r o t e i n ) w e r e i n c u b a t e d w i t h 100 ~M [1-14C]PFDA a s d e a c r i b e d i n M a t e r i a l s a n d M e t h o d s . R e s u l t s a r e m e a n ± S . E . (n = 4). Prelmration

Cofactors

Covalently bound PFDA (pmol/mg protein)

ATP

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142 ± 25 a 84 ± 47 a 7 9 ± 41 a

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245 ± 48 b 218 • 16 b

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31 a 136 ± 17 a 132 ± 9 a

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149

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Microsomal preparations containing covalently bound [1-14C]PFDA were washed with organic solvents as described above, the protein pellet resuspended in a small volume of NaOH (0.1 M) and subsequently applied to a silica gel thin layer chromatographic (TLC) plate. The chromatographic behavior of the micro: somal radioactivity was compared to that of authentic [1-14C]PFDA in a system used to detect PFDA metabolites [12]. The PFDA-derived radioactivity associated with protein remained at the origin and did not migrate similarly to the parent compound (data not shown). Therefore, the radioactivity associated with protein does not represent contamination of sample with unbound PFDA and provides further evidence for the covalent association of PFDA with microsomal proteins. DISCUSSION

These studies were instigated to determine if perfluorinated acids are able to covalently bind to proteins. Protein samples were exhaustively extracted with organic solvents until radioactivity could no longer be detected in the wash. Thus covalent binding of xenobiotics to proteins is defined as suggested by Pohl and Branchflower [13]. Despite the metabolic inertness of the perfluorinated fatty acids, the results indicate that PFDA and PFOA bind to proteins in the plasma,

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liver and testes in a covalent manner. Although PFDA- and PFOA-derived radioactivity could be found covalently associated with protein following the organic extractions,the amount of radioactivityassociatedwith any given protein was not enough to be seen following SDS-PAGE/autoradiography. The in vitro covalent binding of P F D A and P F O A could be reduced upon the addition of cysteine but not methionine to the incubation. This suggests, although does not prove, that protein sulfhydrylgroups are the siteof covalent attachment of P F D A and PFOA. However, since other thiol containing compounds such as G S H and C o A had no effect on the covalent binding of P F D A to microsomal or cytosolicproteins,a sulfhydrylgroup alone is not sufficientto reduce P F D A covalent binding to protein. In addition, a perfluorinated acidamino acid or -GSH conjugate was not detected in plasma, urine, or bileof male and female rats followingin vivo treatment [4,5].Therefore the role of cysteine in decreasing P F D A covalent binding is probably not due to formation of a PFDA-cysteine adduct but involves a more complicated effect,possiblyby protecting protein thiolsby formation of disulfidebonds. Interestingly, a cytosolic component appears to be modulating the in vitro covalent binding of P F D A to microsomes prepared from rat liver.This factor does not appear to be G S H as microsomal P F D A incubations with or without G S H yielded similarcovalent binding of PFDA-derived 14C to protein.This protectiveeffectmay be due to non-covalentbinding of the perfluorinatedfatty acid to proteins,as has been demonstrated for the cytosolicprotein fattyacid-binding protein [14]. The perfluorinatedfatty acids are potent peroxisome proliferatorsin rats.The rodent peroxisome proliferators nafenopin, bezafibrate and M E D I C A 16 covalently bind to specificproteins in cultured rat hepatocytes [15]. Furthermore, sulfur-or oxygen-substitutedfatty acid analogs undergo covalent association with proteins in vitro [16].Lipofuscin accumulation seen in the liverof rats treated with peroxisome proliferators,may be a result of covalently modified proteins accumulating in the lysosome [17]. This correlation lead Hertz and Bar-Tana to suggest that covalent modificationof a specifictarget protein may lead to the peroxisome proliferativeresponse [18].In support of thishypothesis, the present study demonstrates that perfluorinated fatty acids strongly associate with proteins in vitro and in vivo. Whether P F D A and P F O A are covalently attaching to the same proteins as other peroxisome proliferatorsin the livercould not be determined*. Although covalent binding to protein is not always proportional to the toxicity of a xenobiotic,itdoes indicatethat a reactivemetabolite has been formed [19].

*In preliminary experiments we saw an association of P F D A and P F O A with several proteins in hepatocyte suspensions and in primary hepatocytes in culture following separation with SDS-PAGE [27].However, in future experiments, despite detecting covalent binding of P F D A and P F O A to proteins in culture (0.4 pmol/mg protein), we could not detect radioactive bands in the gel as before. It was later determined that the 9 - 10-kDa radioactive band seen previously was the parent compound and could be removed from the gel by soaking in methanol/acetic acid (40o70/10%) for 12 h. The identificationof PFDA- and PFOA-covalently modified proteins is currently being investigated.

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The acylation of proteins via both endogenous and xenobiotic carboxylates may require the formation of an activated CoA ester. Indeed, many peroxisome proliferators appear to be converted to CoA thioesters [19]. Nonetheless, the perfiuorinated acids are not metabolized to a CoA conjugate to any appreciable extent. That is, under several conditions where formation of clofibroyl-CoA could be readily demonstrated, no PFDA- or PFOA-CoA was obtained [20]. The fact that PFDA and PFOA could covalently bind to hemoglobin and albumin as well as liver cytosol and microsome preparations in the absence of CoA or ATP suggests that the perfluorinated acids are capable of covalently binding to proteins without first being activated to a CoA thioester. Due to the stability of the carbon-fluorine bond [21] the most probable site of attachment to proteins is via the carboxyl terminal. The electron withdrawing ability of the fluorine atoms in the aliphatic portion of the perfluorinated fatty acids results in stabilization of the anion formed by the dissociation of the carboxyl hydrogen [22]. As a result of these unique physio-chemical properties, the carboxyl terminal of PFDA or PFOA may be in a sufficiently active state to attach to protein sulfhydryl groups whereas endogenous fatty acids and xenobiotic carboxylates (such as other peroxisome proliferators) require prior activation to a CoA conjugate. The role which covalent binding of perfluorinated fatty acids to proteins plays in the mechanism of action of these chemicals is not known. However, since PFDA and PFOA covalently attach to proteins in target organs for perfluorinated fatty acid toxicity, the liver and testes, the possibility that this effect contributes to hepatic and testicular toxicity cannot be discounted either. The possible correlation between covalent binding of peroxisome proliferators to proteins has been suggested. However, since dietary conditions such as vitamin E or riboflavin deficiency also lead to an increase in the number of peroxisomes in rodent liver, the relationship between covalent binding of PFDA, PFOA and other peroxisome proliferators to proteins and the induction of peroxisomes may not be causally related. Based on the low concentrations of covalently bound PFDA or PFOA, it is also unlikely that covalent binding of perfluorinated fatty acids to hepatic proteins results in the altered lipid metabolism seen in rats [1,2]. The testis contains a high relative concentration of PFDA and PFOA covalently bound to proteins; to our knowledge this is the first report of covalent binding of a peroxisome proliferator to proteins in the rat testis. PFDA-treatment results in testicular necrosis and calcification [23] as well as a decrease in steroidogenesis [24] in rats. Although these compounds may not be hepatocarcinogenic [2,25], there is an increase in Leydig cell adenomas in rats treated with PFOA [26]. Therefore, it is possible that the high relative concentration in the testis is correlated with the toxicity observed, especially if the Leydig cells contain a significant portion of the covalently bound perfluorinated fatty acid in the testis. ACKNOWLEDGEMENTS

This study was supported by NIH grant GM41131. Contribution 251, Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706. J.P.

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Vanden Heuvel was supported by NIEHS training grant ES07015 awarded to the Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin. REFERENCES 1 M.J. Van Rafelghem, J.P. Vanden Heuvel, L.A. Menahan and R.E. Peterson, Perfluorodecanoic acid and lipidmetabolism in the rat, Lipids, 23 (1988) 671- 678. 2 T.P. Pastoor, K.P. Lee, M.A. Perri and P.J. Gillies,Biochemical and morphological studies of ammonium perfluorooctanoate-inducedhepatomegaly and peroxisome proliferation,Exp. Mol. Pathol., 47 (1987) 98-109. 3 M.J. Van Rafelghem, D.R. Mat-tie,R.H. Bruner and M.E. Andersen, Pathologicaland hepatic ultrastructuraleffectsof a single dose of perfluoro-n-decanoicacid in rat, hamster, mouse and guinea pig, Fund. Appl. Toxicol., 9 (1987) 522-540. 4 J.P. Vanden Heuvel, B.I. Kuslikis, M.J. Van Rafelghem and R.E. Peterson, Disposition of perfluorodecanoicacid in male and female rats,Toxicol.Appl. Pharmacol., 107 (1991)450- 459. 5 J.P.Vanden Heuvel, B.I. Kuslikis,M.J, Van Rafelghem and R.E. Peterson, Tissue distribution, metabolism and eliminationof perfluorooctanoicacid in male and female rats,J. Biochem. Toxicol.,6 (1991) 83-92. 6 M.E. George and M.E. Andersen, Toxic effectsof nonadecafluoro-n-decanoicacid in rats,Toxicol.Appl. Pharmacol., 85 (1986) 169-180. 7 M. Ylinen and S. Auriola, Tissue distributionand eliminationof perfluorodecanoic acid in the rat after a single intraperitonealadministration,Pharmacol. Toxicol.,66 (1990) 45-48. 8 M. Ylinen, H. Hanhijftrvi,J. Jaakonaho and P. Peura, Stimulation by oestradiolof the urinary excretion of perfluorooctanoicacid in the male rat, Pharmacol. Toxicol.,65 (1989) 274-277. 9 I.L. Reich, H.J. Reich, L.A. Menahan and R.E. Peterson, Synthesis of 14C-labeledperfluorooctanoic and perfluorodecanoic acids;purificationof perfluorodecanoic acid, J. Lab. Compds. Radiopharm., 24 (1987) 1235-1244. 10 A. Wollenberger, O. Ristau and G. Schoffa, Eine einfache technik der extrem schnellen abkfihlung grSi~erergewebestQcke, PflfigersArch., 270 (1960) 399- 412. 11 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 12 J.P. Vanden Heuvel, M.J. Van Rafelghem, L.A. Menahan and R.E. Peterson, Isolationand purificationof perfluorodecanoicand perfluorooctanoicacids from rat tissues,Lipids,24 (1989) 526 531. 13 L. Pohl and R.V. Branchflower, Covalent binding of electrophilicmetabolites to macromolecules, Methods Enzymol., 77 (1981) 43-50. 14 J.P.Vanden Heuvel and R.E. Peterson, Effects of perfluorodecanoic acid on lipidmetabolism in primary rat hepatocyte cultures, Fed. Am. Soc. Exp. Biol. J., 5 (1991) Al169. 15 R. Hertz and J. Bar-Tana, The acylationof proteins by xenobioticamphipathic carboxylicacids in cultured rat hepatocytes, Biochem. J., 254 (1988) 39-44. 16 J.G. Conway, R.C. Cattley, J.A. Popp and B.E. Butterworth, Possible mechanisms in hepatecarcinogenesis by the peroxisome proliferatordi(2-ethylhexyl)phthalate,Drug Metab. Rev., 21 (1989) 65-102. 17 R.O. Heuckeroth, L. Glaser and J.I.Gordon, Heteroatom-substituted fatty acid analogs for Nmethyltransferase: An approach for studying both the enzymology and function of protein acylation, Proc. Natl. Acad. Sci. USA, 85 (1988) 8795-8799. 18 J. Bar-Tana, S. Ben-Shoshan, J. Blum, Y. Migron, R. Hertz, J. Pill,G. Rose-Khan and E-C. Witte, Synthesis and hypolipidemic and antidiabetogenic activitiesof ~,~,~',~'-tetrasubstituted, long-chain dioic acids, J. Med. Chem., 32 (1989) 2072-2084. 19 M. Bronfman, Activation of hypolipidaemic drugs to acyl-coenzyme A thioesters,Biochem. J., 239 (1986) 781- 784. 20 B.I. Kuslikis,J.P. Vanden Heuvel and R.E. Peterson, Lack of evidence for perfhorodecanoylor perfluorooctanoyl-coenzyrneA formation in male and female rats, Toxicologist,11 (1991) -

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328 21 H.G. Bryce, in J.H. Simons (Ed), Fluorine ChemiStry, Voh V, Academic Press, New York, 1964, pp. 295- 498. 22 R.A. Pyter, Microenvironmental nonideality effects in adsorption of surfactants and micellar solubilization, Ph.D. Thesis, University of Wisconsin, Madison, 1980, pp. 238- 259. 23 C.T. Olson and M.E. Andersen, The acute toxicity of perfluoreoctanoic and perfluorodecanoic acids in male rats and effects on tissue fatty acids, Toxicol. Apph Pharmacol., 70 (1983) 362- 372. 24 R.C. Bookstaff, R.W. Moore, G.B. Ingall and R.E. Peterson, Androgenic deficiency in male rats treated with perfluorodecanoic acid, Toxicol. Appl. Pharmacol., 104 (1990) 322-333. 25. T. Borges, H.P. Glauert, R.E. Peterson, H.C. Pitet and L.W. Robertson, Lack of promoting activity of perfluorodecanoic acid (PFDA) in 2-stage diethylnitrosamine (DEN)-induced hepatecarcinogenesis, Toxicologist, 11 (1991) 185. 26 M.E. Hurtt, S.M. Murray, S.R. Frame and J.C. Cook, Investigation of a hormonally-mediated mechanism for ammonium perfluorooctanoate (C8)-induced Leydig cell adenomas, Toxicologist, 10 (1990) 192. 27 R.E. Peterson, J.P. Vanden Heuvel and B.I. Kuslikis, Acylation of cellular proteins by perfluorinated fatty acids, Toxicologist, 11 (1991) 185.