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The Peroxisome Proliferator Perfluorodecanoic Acid Inhibits the Peripheral-Type Benzodiazepine Receptor (PBR) Expression and Hormone-Stimulated Mitochondrial Cholesterol Transport and Steroid Formation in Leydig Cells¹

Division of Hormone Research, Departments of Cell Biology, Pharmacology & Neuroscience, Georgetown University Medical Center, Washington, DC 20007

Address all correspondence and requests for reprints to: Vassilios Papadopoulos, Georgetown University Medical Center, Department of Cell Biology, 3900 Reservoir Road, Northwest, Washington, D.C. 20007. E-mail: papadopv{at}gunet.georgetown.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
IT HAS BEEN now well established that in many rodent and nonrodent species, including primates, a massive increase in both the size and number of peroxisomes can be induced by a wide range of xenobiotic compounds named peroxisome proliferators (1, 2, 3, 4). Peroxisome proliferators are a large class of structurally diverse industrial and pharmaceutical chemicals with little obvious similarity except for the presence of an aromatic ring, carboxylic acid, and aliphatic chain. Examples include the widely used fibrate hypolipidemic drugs, certain phthalate ester plasticizers, herbicides, and perfluorinated carboxylic acids, such as the perfluorodecanoic acid (PFDA), that are used in industry as lubricants, surfactants, plasticizers, wetting agents, and corrosion inhibitors (1, 4, 5, 6). All peroxisome proliferators elicit remarkably similar responses in livers that include changes in hepatic peroxisome proliferation, proliferation of liver cells, and hepatocarcinogenesis. Concurrent with the marked increase in number and size of peroxisomes, there is a transcriptional induction of the enzymes responsible for ßoxidation of fatty acids, including acyl coenzyme A oxidase, bifunctional enzymes, and 3-ketoacyl-CoA thiolase, the cytochrome P450 CYP4A enzyme, the fatty acid- and acyl-CoA-binding proteins (2, 3, 4). More recently, peroxisome proliferators were found to regulate the expression of rat malic enzyme (7) and human transferrin genes (8). These morphological and biochemical changes are associated with alterations in lipid metabolism, as reflected by lower plasma triglyceride and cholesterol levels in the treated animals (6, 9, 10). Thus, the transcriptional and morphological changes seen reflect an adaptive response that maintains the homeostasis of cellular lipids. Despite the prevalent use of these compounds, it is only recently that a receptor-based mechanism for their pleiotropic effects was presented with the cloning of the peroxisome-proliferator-activated receptor (PPAR) (11). PPAR is a member of the family of steroid-nuclear receptor proteins and exists in at least three isoforms encoded by separate genes, , ß/, and (3).

In addition to their effect on the liver, peroxisome proliferators also induce pathological changes in a number of target organs, including the testis. In 1983, Olson et al. reported that the testis is a target organ for the 10-carbon perfluorinated carboxylic acid PFDA (perfluorodecanoic acid; nonadecafluoro-n-decanoic acid; CF₃(CF₂)₃COOH) toxicity in the rat, hamster and guinea pig (11, 12). In the rat testis, early degenerative changes first seen 8 days after PFDA treatment progressed to tubular atrophy, calcification, and necrosis by 16 days (12). In 1988, Reddy and co-workers reported that treatment of rats with the peroxisome proliferator ciprofibrate resulted in increased levels of peroxisomal ß-oxidation and catalase genes in the Leydig cells of the testis (13). No changes in the peroxisomal volume or activities were shown in the seminiferous tubule compartment of the testis. More recently, Peterson and colleagues reported that PFDA treatment of rats markedly decreased plasma testosterone and DHT levels with an ED₅₀ of 30 mg/kg (14). Associated with the decrease in plasma testosterone levels were decreased accessory organ weights due to atrophic changes characteristic of decreased plasma androgen concentrations. PFDA had no effect on accessory organs when the androgenic deficiency was prevented by castrating and implanting PFDA-treated rats with testosterone. In these studies, rats were dosed with PFDA and then killed 7 days later. Moreover, PFDA treatment did not affect plasma clearance of testosterone and plasma LH levels, suggesting a direct effect on testicular androgen production. Decapsulated testes from PFDA-treated rats incubated with hCG secreted far less testosterone into the medium that did the control testis (14). Thus, these data present direct evidence that the peroxisome proliferator PFDA targets specifically the interstitial Leydig cell of the testis where it may inhibit the hormone-stimulated steroidogenesis. In addition, Wahli and co-workers recently reported that the Leydig cells and the ovarian granulosa and theca cells express the PPAR and PPARß proteins (15). PPAR and PPARß were also found in Sertoli cells but not in the germ cell line. These findings collectively indicated that the peroxisome proliferator PFDA, probably acting via PPAR inhibits the hormone-stimulated Leydig cell steroidogenesis. In this manuscript, we examined the effect of PFDA on Leydig cell function. We identified the outer mitochondrial membrane peripheral-type benzodiazepine receptor (PBR) protein to be the target of PFDA. PFDA-induced decrease in PBR levels leads to decreased cholesterol transport and steroid synthesis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Purified hCG (batch CR-125 of biological potency 11900 IU/mg) was a gift from NIH. [³H]PK 11195 (Sp. Act., 86.9 Ci/mmol), [-³²P]dCTP (Sp. Act., 3000 Ci/mmol), [³²P]NAD (Sp. Act., 800 Ci/mmol) and [1,2,6,7-N-³H]progesterone (Sp. Act., 94.1 Ci/mmol) were obtained from NEN Life Science Products (Wilmington, DE). PK 11195 and aminoglutethimide were obtained from Research Biochemicals International, Inc. (Natick, MA). Nitrocellulose (0.45 µm) was from Hoefer Scientific (San Francisco, CA). Trilostane (4,5-epoxy-17-hydroxy-3-keto-5-androstan-2-carbonitrile) was donated by Sterling-Winthrop (Surrey, UK). 22R-hydroxycholesterol, dibutyryl cyclic AMP and PFDA were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Cell culture supplies were purchased from Life Technologies, Inc.(Grand Island, NY) and cell culture plasticware was from Corning, Inc. (Corning, NY). Electrophoresis reagents and materials were supplied from Bio-Rad Laboratories, Inc. (Richmond, CA). All other chemicals used were of analytical grade and were obtained from various commercial sources.

Cell culture and treatments
MA-10 mouse Leydig tumor cells were a gift from Dr. Mario Ascoli (University of Iowa) and were plated at low density for 24 h in modified Waymouth’s MB 752/1 medium containing 20 mM HEPES, 1.2 g/liter NaHCO₃, and 15% horse serum, pH 7.4 (16). After 24 h the media were changed and fresh media containing the indicated amounts of PFDA or its solvent propylene glycol (PEG) were added for 24, 48, or 72 h. At the end of the incubation the cells were washed with serum-free medium and stimulated for 2 h with saturating amounts of hCG (1 nM) in serum-free medium. This concentration of hCG was previously shown (16) to maximally stimulate steroid synthesis by these cells. At the end of the 2-h incubation period the cell media were saved for progesterone determination, and the cells were either dissolved in 0.1 N NaOH for protein determination or processed for protein (Western) or messenger RNA (mRNA) (Northern) blot analysis or other biochemical assays.

Testicular interstitial cells were prepared by collagenase dissociation (17) of testes obtained from adult Sprague Dawley (300 g) rats. This preparation contained 20–30% 3ß-hydroxysteroid deshydrogenase positive cells (Leydig cells). Leydig cells were further purified using discontinuous Percoll gradient centrifugation as previously described (17). The preparations obtained contained 75–85% Leydig cells as shown by the histochemical staining for 3ß-hydroxysteroid deshydrogenase (17). Isolated rat Leydig cells were cultured for 48 h with various concentrations of PFDA under the same conditions as described above for the MA-10 cells. At the end of the incubation the cells were washed with serum-free medium and stimulated for 2 h with saturating amounts of hCG (1 nM) in serum-free medium. Media were collected for testosterone measurements.

Measurement of cholesterol transport in mitochondria
For these studies, MA-10 cells cultured in 150-mm culture dishes were treated as described above for 48 h with or without the indicated concentrations of PFDA. At the end of the incubation cells were washed with serum-free medium and treated with 1 nM hCG in the presence of 0.76 mM aminoglutethimide, a specific inhibitor of the P450scc. At the indicated time points, media were removed, the cells were resuspended in PBS and mitochondria were isolated in buffers containing aminoglutethimide as described previously (18). The rate of pregnenolone formation, reflecting the amount of cholesterol available to the P450scc, was determined as previously described in aminoglutethimide-free buffer (18). To examine the cytochrome P450 side chain cleavage activity (P450scc), mitochondria from control and PFDA-treated cells were incubated with 22R-hydroxycholesterol in the presence of 1 µM trilostane, an inhibitor of 3ß-hydroxysteroid dehydrogenase enzyme (16, 19). Pregnenolone formation was measured as an index of the P450scc activity.

RIAs
Progesterone, testosterone and pregnenolone were measured by means of RIA (16, 18) using antibodies specific for each steroid (ICN Pharmaceuticals, Inc., Costa Mesa, CA). The data were analyzed using the MultiCalc software from EGG-Wallac, Inc. (Gaithersburg, MD).

Electron microscopy and morphological analysis
MA-10 cells were plated at low density and after 48 h, the media were replaced with fresh medium alone (control cells) or containing the indicated amounts of PFDA (10^-5 to 10^-7 M) and further incubated for 24, 48, or 72 h. At the end of incubation, both cell groups (control and PFDA-treated) were washed with serum-free medium and then stimulated for 2 h with 1 nM hCG in serum-free medium. Cells were processed for electron microscopic morphometry as we previously described (20). Briefly, cells were fixed in 2% glutaraldehyde and 1% paraformaldehyde solution prepared in 3 mM phosphate buffer (pH 7.4) at 4 C for 20 min, washed with buffer A (3 mM phosphate buffer, pH 7.4) for up to 2 days, and postfixed for 20 min in 1% OsO4 solution mixed with 0.2 M s-collidine/HCl buffer (pH 7.4). The samples were then washed with buffer A, stained in 2% uranyl acetate solution in water for 15 min, washed, dehydrated in upgraded (20–100%) ethanol solutions, embedded in Epon:alcohol mixture 1:1 for 3 h and then in 100% Epon for an additional 3 h, and then overnight in the oven. Thin sections (90–95 nm) of blocks were treated with 1% solution of lead citrate, placed on copper grids, and morphometrically analyzed for the surface density of cytoplasmic lipid inclusions. All observations were made with a JEOL-2000 transmission electron microscope operated at 50–60 kV. Up to seventy sectioned cells, with a cell:nucleus ratio of less than 2:1, from each control and experimental protocol were subjected to morphometric analysis to determine the surface density of cytoplasmic lipids. The analysis of sectioned cellular profiles were made on images magnified X20,000–30,000 with the intersection point count method. Vertical and horizontal separation of the adjacent points was 0.3 µm. The proportion of cellular surface covered with the cytoplasmic lipid inclusions were first established by a simple comparison of numbers of intercept points of test lines coinciding with the inclusions and with the rest of the cell. The surface density value of cytoplasmic lipid pools (Svl) was subsequently calculated as: Svl = (2 x Il)/Lt, where Il is the number of intercepts of test lines with lipid pools, and Lt = 1/2 x Pt x z, where Pt is the number of end points of test lines z (21). This two-dimensional evaluation of the intracellular free lipid content, rather than the three-dimensional assessment of the volumetric presentation of the substrate, was selected by necessity because of the unknown absolute volume of this cellular population.

Analysis of cell viability and mitochondrial integrity
Cell viability was analyzed using the trypan blue exclusion method. At the end of the incubation, the cells were washed three times with PBS and incubated for 15 min with 0.1% trypan blue stain. After washing, stained cells (dead cells) were counted by light microscopy. Cell viability and mitochondrial integrity were also evaluated following the above described cell treatment protocol, by measuring the levels of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction (22) using the TACS MTT proliferation assay (Trevigen, Inc., Gaithersburg, MD).

Total protein labeling
10⁶ MA-10 cells were treated without and with PFDA for 48 h. Cells were then translabeled with ³⁵S-met/cys (0.6 mCi/well) in met/cys free medium for 4 h. Total protein labeling was examined in aliquots precipitated by 10% trichloroacetic acid. The precipitates were heated at 90 C for 20 min, cooled, filtered on glass fiber filters, rinsed with 5% trichloroacetic acid, and washed with ethanol. Filters were air-dried and radioactivity was measured by liquid scintillation spectrometry.

Analysis of DNA damage
The possibility that PFDA may cause DNA damage to the cells was examined using the poly(ADP-ribose) polymerase (PARP) assay (23,24; Trevigen, Inc., Gaithersburg, MD). Cells were collected and resuspended in 50 mM Tris [pH 8.0] buffer supplemented with 25 mM MgCl₂ and 0.1 mM PMSF. Cells were sonicated, centrifuged, and the supernantants were used to determine the PARP activity by measuring the incorporation of radiolabeled poly(ADP-ribose) into their control PARP or cellular PARP. The reaction was stopped using 20% ice-cold TCA and the precipitates were collected on GF/C filters. Filters were air-dried and radioactivity was measured by liquid scintillation spectrometry.

Radioligand binding assays
Radioligand binding assays were performed as previously described (16). In brief, cells were scraped from culture flasks into PBS, dispersed by trituration, and centrifuged at 1200 x g for 5 min. The cells were resuspended in PBS to a final concentration of 5 to 10 µg protein/100 µl. Saturation binding studies were performed on 10 µg protein for the MA-10 cells in a final volume of 300 µl in the presence of the radioligand at the indicated concentrations. Nonspecific binding was determined in the presence of 6 µM of the homologous nonradioactive ligand. After 2 h incubation at 4 C, the assays were stopped by filtration through GF/B filters (Brandel, Gaithersburg, MD) equilibrated in 0.1% polyethyleneimine and washed with 40 ml ice-cold PBS. Radioactivity trapped on the filters was determined by liquid scintillation counting. The dissociation constant (K_d) and the binding capacity were defined following Scatchard analysis of the specific binding using the LIGAND program (25). In some experiments the direct effect of PFDA on PK 11195 ligand binding to isolated MA-10 Leydig cell mitochondria was examined.

SDS-PAGE and immunoblot analysis
Mitochondria were isolated as we previously described (26). Mitochondrial proteins were fractionated by one dimensional SDS-PAGE and electro-transferred onto nitrocellulose (27). The nitrocellulose was subjected to immunoblot analysis using anti-PBR (1:1000) or anti-StAR (1:1000) antisera and goat antirabbit IgG alkaline phosphatase-conjugated secondary antibody followed by the substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate for detection (27). Densitometric analysis of the immunoreactive protein bands was performed using the Sigmagel software (Jandel Scientific, San Rafael, CA). The production and characterization of the anti-PBR and anti-StAR antisera, against internal peptide sequences of the proteins have been previously described (27).

RNA (Northern) blot analysis
Total cellular RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction method (28) using the RNAzol B reagent (Tel-Test Inc., Friendswood, TX). RNA electrophoresis, transfer, probe labeling, and membrane hybridization were performed as previously described (18). The blots were hybridized against the [³²P] complementary DNA (cDNA) probe for PBR or cyclophilin (18). Screen enhanced autoradiography was performed by exposing Kodak X-OMAT AR films to the blots at -80 C for 48 h. Densitometric analysis of the immunoreactive protein bands was performed using the Sigmagel software (Jandel Scientific).

Transient transfections
The MA-10 cells were transiently transfected using the electroporation method. Briefly, each Genepulser cuvette (0.4 cm-gap, Bio-Rad Laboratories, Inc.) contained 8 x 10⁶ cells in 350 µl of antibiotic-free complete Waymouth’s growth medium, plus 50 µl of 0.1 x TE containing the plasmid preparations. The cells were cotrasfected with 30 µg of the pPBR (-1219/+36) CAT construct, and 5 µg per transfection of the pSV promoter-ß-galactosidase plasmid (Stratagene, La Jolla, CA) to check the transfection efficiency. The 1219 bp promoter region fragment contained the first 36 bp of exon 1 of rPBR subcloned into the polylinker of the pCAT-Basic (Promega Corp., Madison, WI) and was provided by Dr. Krueger (29, Georgetown University). As control, MA-10 cells were also transiently co-transfected with 30 µg of the pCAT-basic plasmid (Promega Corp.) and 5 µg per transfection of the pSV promoter-ß-galactosidase plasmid (Stratagene). Cells in electroporation cuvettes were electro-shocked using 330V and a capacitance of 950 µFd generated from Genepulser II (Bio-Rad Laboratories, Inc.). The cells were kept at room temperature for 10 min before plating onto six-well dishes, and further allowed to grow in complete Waymouth’s medium for 16 h. Then, the media were changed into serum-free Waymouth’s and cells were treated with or without 30 µM of PFDA. Twenty-four hours later, the cells were harvested for CAT-ELISA (5-Prime3-Prime Inc., Boulder, CO) and ß-galactosidase (Promega Corp.) activity measurements.

Measurement of PBR mRNA stability
To examine the effects of PFDA on PBR mRNA stability we treated MA-10 cells for 48 h with the indicated concentrations of PFDA and then measured the decay of PBR mRNA by incubating the cells with actinomycin D (10 µg/ml) for an additional 15, 30, 60, and 180 min. Longer exposures to actimomycin D were toxic to the cells. Total cellular RNA was isolated as described above and PBR mRNA levels were measured by RNA (Northern) blot analysis.

Protein measurement
Protein was measured by the method of Bradford (30), using BSA as a standard.

Statistics
Statistical analysis was performed by one-way ANOVA and unpaired Student’s t test using the INSTAT 3.00 package from GraphPad Software, Inc. (San Diego, CA). In the case of morphological analyses the Fisher’s PLSD test was used.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PFDA inhibits the hCG-stimulated Leydig cell steroidogenesis
To examine the chronic effects of PFDA on Leydig cell function, we treated the cells for 24, 48, and 72 h with increasing concentrations of PFDA or the solvent used PEG. The cells were then washed and treated for 2 h with saturating concentrations of hCG. This experimental scheme should define whether long term exposure to PFDA affects the ability of the cells to respond to hormones. Treatment of MA-10 Leydig cells with increasing concentrations of PFDA (10^-7 to 3 x 10^-3 M), induced a 10–95% inhibition of a 2 h hCG-stimulated progesterone biosynthesis, in dose- and time-dependent manner (Fig. 1A). This effect was statistically significant (ANOVA, P < 0.001) at all treatment periods. This inhibitory effect of PFDA is shown to be significant after 24 h of treatment with PFDA at concentrations higher than 3 x 10^-5 M and with an ED₅₀ of 10^-5 M. Treatment of the cells for longer periods of time reduced the ED₅₀ to 3 x 10^-6 M for the 48 h treatment and 10^-6 M for the 72 h treatment. PEG had no effect on the hCG-stimulated progesterone production.

View larger version (14K): [in this window] [in a new window]   Figure 1. Dose-response effect of PFDA on Leydig cell steroid formation. A, MA-10 Leydig cells were cultured in the presence of the indicated concentrations of PFDA for the indicated time periods. At the end of the incubation, cells were washed and incubated with 1 nM hCG in serum-free media for 2 h. Progesterone production was measured in the media by RIA and cells were lysed for protein determination. B, Isolated rat Leydig cells were cultured for 48 h in the presence of the indicated concentrations of PFDA. At the end of the incubation, cells were washed and incubated with 1 nM hCG in serum-free media for 2 h. Testosterone production was measured in the media by RIA. Results shown are means ± SD (n = 9) from three independent experiments. Statistical analysis by ANOVA demonstrated an extremely significant effect of PFDA with a P < 0.001.

The results presented above on the MA-10 mouse tumor cell line were confirmed using isolated adult rat Leydig cells (Fig. 1B). 48 h treatment with PFDA resulted in the inhibition of the subsequent hCG-stimulated testosterone production with and ED₅₀ of 3 x 10^-6 M, similar to that seen in MA-10 cells. These results validate the use of the MA-10 cell model for identifying the mechanism of PFDA inhibitory action on Leydig cell steroidogenesis.

PFDA does not affect Leydig cell viability, mitochondrial integrity, total protein synthesis and DNA
Concentrations of PFDA up to 10^-4 M do not affect cell viability as attested by the trypan bleu exclusion test. However at 3 x 10^-4 M 30–40% of the cells had taken up the trypan blue dye, indicating that at this concentration the cells were dying. These results were validated using the MTT assay. In Fig. 2, the effect of 48 h treatment with increasing concentrations of PFDA on cell cultures where different numbers of cells were plated is shown. It is clear again that PFDA up to 10^-4 M does not affect cell viability and more specifically mitochondrial integrity because the MTT assay is based on the MTT reduction by the mitochondrial diaphorase enzyme, an index of mitochondrial integrity (22).

View larger version (24K): [in this window] [in a new window]   Figure 2. Dose-response effect of PFDA on Leydig cell mitochondrial integrity and cell viability. The indicated numbers of MA-10 Leydig cells were cultured in 96-well plates for 48 h in the presence of the indicated concentrations of PFDA. At the end of the incubation, cells were washed and levels of mitochondrial integrity/cell viability were measured using the MTT assay as described in Materials and Methods. Results shown are means ± SD (n = 6) from two independent experiments each conducted in triplicates.

View larger version (67K): [in this window] [in a new window]   Figure 3. Effect of PFDA on total protein synthesis. MA-10 cells were treated for 48 h with 10 µM PFDA. Cells were then translabeled with 35S-met/cys in met/cys free medium for 4 h. Total protein labeling was determined from acid-insoluble counts and scintillation counting. Results shown are means ± SD (n = 8) from two independent experiments each conducted in quadruplicates.

View larger version (45K): [in this window] [in a new window]   Figure 4. Effect of PFDA on poly(ADP-ribose) polymerase enzyme activity. MA-10 cells were treated for 48 h with 10 µM PFDA or propylene glycol (PEG) used to dissolve PFDA. At the end of the incubation samples were prepared and the poly(ADP-ribose) polymerase enzyme activity, marker of DNA damage, was determined by measuring the incorporation of radiolabeled NAD. Quantitative values were determined from acid-insoluble counts and scintillation counting. Results shown are means ± SD (n = 8) from two independent experiments each conducted in quadruplicates.

View larger version (141K): [in this window] [in a new window]   Figure 5. Morphological analysis of Leydig cells. Cells were treated for 48 h with 10 µM PFDA. At the end of the incubation cells were washed and treated for 2 h in serum-free media with 1 nM hCG. Cell were then fixed and processed for electron microscopy as described in Materials and Methods. (1 ) Intact (micellar configuration) inclusion (indicated by the asterisk) is demonstrated in the periphery of the cells. A cisterna of endoplasmic reticulum (arrows) encircles about three quarters of the inclusion’s circumference and is closely apposed to its surface. Such a relationship between the inclusion and the endoplasmic reticulum is suggestive of a release of phospholipids from the reticular membrane into the phospholipid pool. The content of the inclusion is homogeneous and moderately electron opaque, a typical appearance of phospholipids grouped in a micellar organization. Original magnification 4,000x. (2 ) In the inclusion demonstrated herein phospholipids are beginning to align in a typical bilayer configuration (arrows). Transition from micellar to bilayer grouping of phospholipids is electron microscopically characterized as a homogeneous medium of high electron density (small arrows). Some mitochondria (M) are present in the vicinity of the inclusion. Original magnification 4,000x. (3 ) Cross section of a Leydig MA-10 mouse tumor cell illustrates the required cellular profile for the morphometric analysis. In addition to the usual organellar profiles (nucleus N, mitochondria M, Golgi zone G, reticulum R), this cell contains several lipid/phospholipid inclusions (indicated by the asterisk). In this section all inclusions appear to contain phospholipids organized in micellar, labile mode which, unless immediately metabolized, will assume a stable bilayer configuration. Original magnification 4,000x.

View this table: [in this window] [in a new window]   Table 1. Thirty five to 70 representative sections, from three independent experiments, made approximately at the midnuclear level (Fig. 5.3), for each cellular category were analyzed for the lipid/phospholipid surface density within the cytoplasm and the average values and the standard errors were derived as shown

Table 2 shows that 48 h treatment with 10 µM PFDA inhibits the cAMP-stimulated progesterone production suggesting that the effect of PFDA was localized beyond the cAMP production. We then examined whether PFDA affected the first step in steroid biosynthesis, the transformation of cholesterol into pregnenolone by the P450scc enzyme located within the inner mitochondria membrane. MA-10 cells were treated for 48 h with 10 µM PFDA and then incubated in the presence of 22R-hydroxycholesterol (10 µM), the hydrosoluble form of cholesterol, which enters readily the inner mitochondria membrane. Progesterone production was identical in both control and PFDA-treated cells, suggesting that the site of action of PFDA is not at the P450scc or other steroidogenic enzyme level but rather at an earlier step of the pathway (Table 2). In agreement with these results, mitochondria isolated from control and PFDA-treated MA-10 cells and incubated with 22R-hydroxycholesterol also produced similar amounts of pregnenolone (data not shown). Thus, it seems like the inhibitory effect of PFDA on steroidogenesis may be due to an impairment of the multistep process of cholesterol mobilization, transport into mitochondria and loading onto P450scc in response to hCG stimulation. To determine whether the hCG-induced translocation of cholesterol to the inner mitochondria membrane was affected by PFDA treatment, we treated cells with aminoglutethimide before and during the 2 h hCG stimulation. In this experiment, aminoglutethimide was used to inhibit the transformation of cholesterol to pregnenolone by P450scc, thus we could determine the amount of endogenous cholesterol accumulated within the inner mitochondrial membrane upon hCG stimulation. Fig. 6 shows that upon washing out of the aminoglutethimide from the isolated mitochondria, pregnenolone production by mitochondria isolated from MA-10 cells treated with PFDA was significantly inhibited compared with control cells (P < 0.001 by ANOVA). This data indicates that PFDA inhibits the transfer or loading of cholesterol to the inner mitochondrial membrane P450scc. We then examined the effect of PFDA treatment on the two known proteins shown to be involved in the cholesterol translocation to the inner mitochondria membrane, PBR (31) and the steroidogenic acute hormone regulator protein (StAR; 32).

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of PFDA on cholesterol transport. MA-10 cells were treated with 10 µM PFDA for 48 h. At the end of the incubation cells were washed with serum-free medium and treated with hCG (1 nM) and aminoglutethimide (0.76 mM). At the indicated time points cell media were aspirated and mitochondria were prepared in buffers containing aminoglutethimide. At the end of the preparation the rate of pregnenolone formation was measured in aminoglutethimide-free buffers as described in Materials and Methods. The results shown are means ± SD (n = 9) from three independent experiments each conducted in triplicates. Analysis of the data by ANOVA indicated that the P value is <0.001, considered highly significant.

View larger version (17K): [in this window] [in a new window]   Figure 7. Effect of PFDA on PBR ligand binding. MA-10 cells were treated with the indicated concentrations of PFDA for 48 h. At the end of the incubation and at each PFDA saturation isotherms of PBR ligand binding were performed using [3H]PK 11195 as ligand. Results obtained were analyzed as described in Materials and Methods and the ligand affinity and capacity were determined. The figure shows the PK 11195 ligand capacity as a ligand. The results shown are means ± SD (n = 3) from a representative experiment. Similar results were obtained in three independent experiments. Analysis of the data by ANOVA indicated that the P value is <0.001, considered highly significant.

View larger version (31K): [in this window] [in a new window]   Figure 8. Effect of PFDA on PBR and StAR protein levels. MA-10 cells were treated with 10 µM PFDA or the solvent PEG for 48 h. At the end of the incubation, cells were washed and incubated with 1 nM hCG in serum-free media for 2 h. Mitochondria were isolated from the cells and the levels of the 18-kDa PBR and 30-kDa StAR proteins were determined by immunoblot analyses. Methods for the preparation of mitochondria, immunoblot and image analyses are described in Materials and Methods. Upper and middle panels show representative immunoblots for the 18-kDa PBR and 30-kDa StAR proteins, respectively. Lower panel shows the means ± SD of the densitometric analysis of the PBR and StAR immunoreactive bands from three independent experiments.

View larger version (38K): [in this window] [in a new window]   Figure 9. Effect of PFDA on PBR mRNA levels. MA-10 cells were treated with 10 µM PFDA for 48 h. At the end of the incubation RNA was isolated from the cells and PBR mRNA levels were determined as described in Materials and Methods. Top panel shows the ethidium bromide staining, indicating loading of the samples from a representative experiment. Middle panel shows the corresponding autoradiogram of the mRNA blot. Bottom panel shows the means ± SD of the relative intensity of the 18-kDa PBR mRNA/18S rRNA, determined as indicated in Materials and Methods, from three independent experiments. Controls were treated with propylene glycol (PEG) used to dissolve PFDA.

PFDA does not affect PBR mRNA transcription but accelerates its decay
The results presented above suggest that PFDA exerts a direct effect on PBR mRNA stability or DNA transcription. Preliminary nuclear run-on experiments failed to provide conclusive evidence on the effect of PFDA on PBR mRNA transcription. Thus, we decided to extend these studies by examining the effects of PFDA treatment on PBR gene transcription. It has been shown previously that the -1219/+36 DNA region upstream from the transcription start site of the PBR gene (29), contains the regulatory elements which are necessary and sufficient for the expression of PBR in MA-10 mouse Leydig tumor cells. The eventual role of the promoter elements present within this region implicated in the down-regulation of the PBR gene transcription by PFDA was examined, with transient transfection experiments. The -1219/+36 fragment within the PBR promoter region subcloned into a CAT-reporter plasmid, was used. Promotorless vector was used as internal control. MA-10 mouse Leydig tumor cells transfected with the pPBR (-1219/+36)-CAT construct, were treated with and without 30 µM PFDA. PFDA was dissolved in 50% PEG. Cells treated with the corresponding amounts of PEG were used also as controls demonstrating that PEG did not have any effect either in the cells function or PBR expression. When the transfected cells were treated with PFDA, no significant changes in the expression of CAT activity were observed (Fig. 10).

View larger version (19K): [in this window] [in a new window]   Figure 10. Effect of PFDA on PBR gene transcription in MA-10 mouse Leydig tumor cells. MA-10 mouse Leydig tumor cells were transiently transfected with the reporter construct pPBR (-1219/+36)-CAT, as well as the control plasmid, pCAT-Basic. The cells were then treated for 24 h in serum-free medium with and without 30 µM PFDA or the solvent PEG. CAT activity was measured as described in Materials and Methods. Controls were treated with propylene glycol (PEG) used to dissolve PFDA. The results shown are means ± SD (n = 12) from three independent experiments.

View larger version (44K): [in this window] [in a new window]   Figure 11. Effect of PFDA on PBR mRNA stability. MA-10 cells were treated with 10 µM PFDA for 48 h. At the end of the incubation cells were exposed to actinomycin D for the indicated time periods. Cells were scraped and PBR mRNA levels were analyzed as described in Materials and Methods. Upper panel shows PBR and cyclophilin mRNA levels from a representative experiment. Lower panel shows the PBR mRNA data expressed as percent of the corresponding control treated with PEG, are the means ± SD of two separate experiments (n = 5).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has been shown that the testis is one of the target organs affected by peroxisome proliferators. The peroxisome proliferator perfluorinated carboxylic acid PFDA was reported to induce early degenerative changes that progressed to tubular atrophy (11, 12). The same peroxisome proliferator was also reported to suppress plasma testosterone levels, without affecting plasma LH concentrations, thus inducing a decrease in accessory organ weights (13). Because implants of testosterone reversed the effect of PFDA and testes from PFDA-treated rats were far less responsive to LH than control testes, it can be concluded that this peroxisome proliferator exerts a direct effect on the hormone-stimulated testosterone biosynthesis. The peroxisome proliferators phthalates were also shown to reduce in vivo and in vitro testosterone levels and induce testicular atrophy (34, 35). Moreover, it has been shown that, in testis, only the Leydig cell is a target for a fibrate hypolipidemic drug, also member of the peroxisome proliferator family of chemicals (13). In addition, it was recently reported that peroxisome proliferators inhibit progesterone synthesis by a human choriocarcinoma cell line in culture (36). Thus, these data suggest that peroxisome proliferators target specifically steroidogenic cells, such as the Leydig cell, where they may control the process of steroid synthesis and thus gonadal function.

Indeed, the data presented herein clearly demonstrates that PFDA inhibits in a dose- and time-dependent manner the hCG-stimulated progesterone production by the MA-10 mouse tumor Leydig cells. PFDA inhibited with the same potency the hCG-stimulated testosterone production by isolated rat Leydig cells, indicating that the results seen with the MA-10 cells were not specific to a tumor Leydig cell line. PFDA also inhibited the cAMP-stimulated progesterone synthesis by MA-10 cells, but did not affect the 22R-hydroxycholesterol-supported steroid formation by these cells. 22R-hydroxycholesterol is an hydrosoluble form of cholesterol, which enters readily the inner mitochondria membrane, and an intermediate of the P450scc reaction (37). This result suggests that PFDA did not act on the P450scc or other steroidogenic enzymes involved in the biosynthesis of the final steroid product. Moreover, these data, together with the reversibility data, suggest that treatment with PFDA for times up to 48 h does not exert a toxic effect on Leydig cells. These results were corroborated by a series of assays performed to assess the potential toxic effect of PFDA on mitochondrial integrity, total protein synthesis, DNA, and cell viability. All tests performed clearly showed that PFDA, under the conditions, concentrations and treatment time periods used, does not exert any toxic effect on the cells. Moreover, a detailed morphologic analysis of the PFDA-treated cells failed to demonstrate any morphological alterations of the cells, compared with control, with one exception, the state of the lipid/phospholipid configuration in the lipid droplets of the cells. In conclusion, the peroxisome proliferator PFDA exerts a specific antisteroidogenic effect on Leydig cells. If this effect of peroxisome proliferators is conserved among species, concern is raised about the risk in human populations exposed for long term to peroxisome proliferators. Thus, characterization of the molecular determinants involved in the response of Leydig cells to peroxisome proliferators such as PFDA will be useful in developing a predictive model of risk to humans.

Because PFDA did not directly affect P450scc enzyme activity, it was clear that the steroid biosynthetic pathway was not affected by the treatment but it was rather the substrate (cholesterol) availability, which it was limiting. The rate-determining step in steroid biosynthesis, however, is not the rate of metabolism of cholesterol to pregnenolone by the P450scc but rather the transport of the precursor cholesterol from intracellular stores to the inner mitochondrial membrane where the P450scc is located (38, 39). Morphologic analysis of the cells treated with and without PFDA indicated that the treatment induced a build-up of cytoplasmic lipid/phospholipid pools that could be due to either reduced utilization and/or release from the cell of this substrate. PFDA-treated cells appeared to have a faster rate of transition of lipids/phospholipids from the micellar to bilayered configuration than the corresponding control cells. Consequently, the treated cells contained significantly greater amount of bilayer-configured lipids/phospholipids in the cytoplasm than control cells. Because hCG has been shown to reduce the number of lipid droplets in Leydig cells in vivo (40) in a time-frame close to the hCG-induced steroid production, it has been suggested that cholesterol esters form, at least part, of the content of these lipid droplets. De-esterified cholesterol from these lipid droplets is part of the substrate used for testosterone formation by Leydig cells. The fact that the transitional configuration of substrate, the only configuration released from the cell (20), was found in the greater quantity in the PFDA-treated cells suggests that most of the cytoplasmic substrate was not transported to the mitochondria and instead accumulated in the droplets. These results further suggest that PFDA’s site of action is at the level of cholesterol transport into the mitochondria.

Biochemical studies using the inhibitor of P450scc aminoglutethimide, which allows for the accumulation of transported cholesterol into the inner mitochondrial membrane by blocking its further metabolism, followed by release of the inhibition and measurement of the synthesized pregnenolone, demonstrated that indeed PFDA inhibited the transfer of cholesterol to the P450scc. Because PFDA did not affect the Leydig cell mitochondrial integrity, we then examine its effect on the expression of molecular entities involved in cholesterol transport in steroidogenic cells. As we noted earlier, two such regulatory elements of the steroidogenic machinery have been identified: PBR (31) and StAR (32).

PBR, although present in all tissues examined, was found to be particularly high in steroid producing tissues, where it was primarily localized in the outer mitochondrial membrane (31). Numerous in vitro and in vivo studies demonstrated that PBR is a functional component of the steroidogenic machinery (16, 31, 41, 42, 43) mediating cholesterol delivery from the outer to the inner mitochondrial membrane (26). An 18-kDa isoquinoline-binding protein was identified as PBR, cloned and expressed (31, 41). Strong evidence was presented suggesting that PBR is a multimeric complex (41) located at the junctions between outer and inner mitochondrial membranes (contact sites) where the PBR complex could function as a pore, thus allowing the translocation of cholesterol from the outer to the inner mitochondrial membrane (44). Further support for this hypothesis was provided by first, molecular modeling studies of the human and mouse 18-kDa PBR protein where it was clearly shown that PBR is a cholesterol channel (44, 45) and second, in vitro reconstitution studies in bacteria where the expression of the 18-kDa PBR protein induced cholesterol transport into these cells (46). We demonstrate herein that a decrease in PBR expression after treatment with PFDA results in reduced hCG-stimulated steroid formation by Leydig cells. The cause-effect relation between the effect of PFDA on PBR expression and steroid synthesis was further supported by the observation that both PBR levels and steroid synthesis returned to normal upon the end of the treatment.

Thus, we focused our studies on examining the effect of PFDA on PBR. We did not see any direct effect of PFDA on PBR ligand binding studies, which excludes the possibility that this compound may act as a PBR ligand. We then demonstrated that PFDA reduced Leydig cell PBR ligand binding, protein, and mRNA levels, resulting in reduced ability of the cells to synthesize progesterone in response to hCG. Although it is difficult to quantify accurately PBR protein and mRNA levels, the comparison of the PFDA-induced reduction of steroid formation with PBR ligand binding, 18-kDa PBR protein, and mRNA levels, as determined by image analysis, shows a close correlation between the message, protein, activity, and function. This observation further supports the cause-effect relation between PBR levels and the ability of Leydig cells to form steroids in response to hCG.

It has been shown that the 30-kDa StAR is expressed in response to trophic hormones, in parallel with steroid production, and it has been suggested to be the key mediator of the hormone-stimulated steroidogenesis (32, 47). No changes in hCG-induced StAR levels were observed upon treatment of the cells with PFDA. Considering that, in the in vivo treatment studies with PFDA no changes in circulating LH levels were reported (14), these data suggest that the antiandrogenic effect of PFDA was not due to an effect on the hormone-induced 30-kDa StAR levels.

The regulation of PBR levels by a number of factors, including hormones and stress, has been reported (31, 48, 49) and more recently both positive and negative regulatory transcriptional elements have been identified in the sequence upstream the first exon of the rat PBR gene (29). Thus, we can speculate that PFDA may control the expression, affect the PBR mRNA stability, or regulate the activity of one or more of these transcriptional elements.

Peroxisome proliferator-responsive genes were found to contain PPREs consisting of a near-perfect direct repeat (DR) of two copies of the sequence TGA/TCCT separated by one bp (DR-1). However, only half of the nucleotides within each repeat sequence are strictly conserved, whereas others vary considerably (3, 8, 50). A fragment spanning from 36 bp into the first exon of PBR with 1,219 bp of upstream genomic sequence has been isolated (29). This upstream sequence was analyzed for transcriptional promoter activity and revealed the presence of three regulatory elements. A closer look at the negative acting regulatory element indicated the presence of a 14-bp sequence with high homology to the previously reported PP response elements (PPREs), suggesting that the molecular determinant required for PP action on gene expression is present in the promotor sequence of the PBR gene. Moreover, recent publications identified peroxisome proliferator activated receptor immunoreactive proteins (PPAR and PPARß) in the Leydig cells of the testis and the granulosa and theca cells of the ovary (15, 51). In addition, it is known that Leydig cells respond to retinoids, suggesting that they may express the retinoic X receptor (RXR). The dimerization partner RXR (3) potentiates gene regulation by PPAR. Taken together these results suggest that indeed the antiandrogenic effect of the peroxisome proliferator PFDA may be due to a direct effect on PBR mRNA expression.

In vitro transfection studies of the PBR promotor into Leydig cells followed by PFDA treatment of the cells indicated that PFDA did not affect the PBR gene expression directly. However, we cannot exclude the possibility that PFDA may have a small effect on PBR expression, which is not obvious, because it overlaps with the effect of other positively regulated elements in the PBR promotor. Further studies on PBR mRNA stability indicated that PFDA might act by accelerating the PBR mRNA decay.

Considering the potential health risk issue associated with PFDA toxicity the question arises whether the levels required to see the effect of PFDA on PBR mRNA levels and steroidogenesis in vitro are likely to be achieved in vivo. To answer this question one should consider that 1) in vivo dosing of the rats with PFDA for 7 days decreased plasma T with an ED₅₀ of 30 mg/kg (13); and 2) the in vitro 48 h treatment with PFDA inhibited the hormone-stimulated steroidogenesis with ED₅₀ of 10 µM (5.14 mg/liter). Considering the blood volume of the adult rat, and without taking into account the PFDA tissue distribution, pharmacokinetics and excretion, we can estimate that the in vitro system is at least 6-fold more sensitive than the in vivo model and provides a sensitive system to test for the effects of PFDA. In addition, the facts that 1) organic fluorine, derived from perfluorocarboxylic acids, persists in the blood of industrial workers exposed to these substances (13), 2) in industrial areas the density of peroxisome proliferators is high (52); and 3) the levels of these compounds in food samples reach the levels of 20 mg/kg (53) indicate that PFDA, as all peroxisome proliferators, is a putative health hazard.

In conclusion, PFDA is shown to inhibit the expression of the PBR protein, which controls the amount of cholesterol transported to the P450scc and consequently the amount of steroids formed by Leydig cells. Ultimately, these changes will affect spermatogenesis (14) and fertility. Industrial and pharmaceutical peroxisome proliferator compounds, some of which are extensively used in humans, may exert similar antiandrogenic effects by inhibiting PBR expression, cholesterol transport, steroid biosynthesis and hence testicular function. Whether the effect of PFDA on Leydig cell PBR expression is unique or representative of other classes of peroxisome proliferators is under investigation.

	Acknowledgments

 
We would like to thank Dr. M. Ascoli (University of Iowa, IA) for the MA-10 Leydig cell line, Dr. K. E. Krueger (Georgetown University, Washington, DC) for the rat PBR promotor, the National Hormone and Pituitary Program (NICHD, NIH) for the hCG, and Sterling-Winthrop (Surrey, UK) for the trilostane.

	Footnotes

 
¹ This work was supported by Grant R01-ES-07747 from the National Institutes of Health.

² Current address: Endocrinologie Moléculaire de la Reproduction, UPRES-A CNRS 6026, Université de Rennes I, 35042 Rennes Cedex, France.

³ Current address: Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.

Received March 23, 2000.

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