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a Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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isomer of hexachlorocyclohexane (HCH), is one of the oldest synthetic pesticides still in use worldwide. Numerous reports have shown that this pesticide adversely affects reproductive function in animals. Although the pathogenesis of reproductive dysfunction is not yet fully understood, recent reports indicate that lindane can directly inhibit adrenal and gonadal steroidogenesis. Because Leydig cells play a pivotal role in male reproductive function through the production of testosterone, the mouse MA-10 Leydig tumor cell line was used to assess the potential effects of -HCH and its isomers, 
-HCH and 
-HCH, on steroid production, steroidogenic enzyme expression and activity, and steroidogenic acute regulatory (StAR) protein expression. StAR mediates the rate-limiting and acutely regulated step in hormone-stimulated steroidogenesis, the intramitochondrial transfer of cholesterol to the P450scc enzyme. Our studies demonstrate that -, -, and -HCH inhibited dibutyryl ([Bu]2) cAMP-stimulated progesterone production in MA-10 cells in a dosage-dependent manner without affecting general protein synthesis; and protein kinase A or steroidogenic enzyme expression, activity, or both. In contrast, each of these isomers dramatically reduced (Bu)2cAMP-stimulated StAR protein levels. Therefore, our results are consistent with the hypothesis that -, -, and -HCH inhibited steroidogenesis by reducing StAR protein expression, an action that may contribute to the pathogenesis of lindane-induced reproductive dysfunction.
Leydig cells, male sexual function, signal transduction, stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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isomer, being the only isomer that possesses significant insecticidal activity. HCH has been used extensively to control malaria, but is more commonly used to eradicate insects in agriculture and to treat lice infestation in humans, poultry, and livestock. Because of its widespread use and chemical stability, HCH has become widely distributed in ecosystems and is now a global pollutant. Several studies have demonstrated that lindane disrupts reproductive function in male and female animals. In male rats, chronic exposure to lindane markedly reduces serum testosterone levels, epididymal sperm counts, and sperm motility, whereas in guinea pigs, it damages seminiferous tubules and completely arrests spermatogenesis [1–5]. Similarly, in female mice and rabbits, lindane reduces serum estrogen and progesterone levels, whereas in pregnant mice and minks, it decreases whelping rate and litter size [6–10]. Exposure to lindane during the first 4 days of pregnancy completely prevented implantation in mice, but normal pregnancy resulted when estrogen and progesterone were coadministered with lindane [8]. Because adequate levels of testosterone are required for normal spermatogenesis, and estrogen and progesterone are required for pregnancy, a decline in serum steroid hormone levels likely contributes to the pathogenesis of lindane-induced infertility.
Several studies indicate that lindane can directly inhibit steroid hormone biosynthesis. First, following exposure in vivo, -HCH and its isomers concentrate in ovary and testis, the sites of steroid hormone production [1, 11]. Second, lindane reduces serum hormone levels at concentrations that fail to cause Leydig cell degeneration in vivo or in vitro, indicating that this pesticide may directly target components of the steroidogenic pathway [1–5]. Finally, providing direct evidence that lindane inhibits steroid production, lindane and its isomers, -HCH and -HCH block the conversion of cholesterol to pregnenolone in steroidogenic cells in vitro without affecting P450scc activity [2, 6, 12]. This latter finding indicates that these compounds may inhibit steroidogenesis by interfering with the availability of cholesterol to this enzyme, and is significant because the supply of cholesterol to the P450scc enzyme constitutes the true rate-limiting step in steroid hormone biosynthesis.
Considerable evidence indicates that the steroidogenic acute regulatory (StAR) protein mediates the rate-limiting and acutely regulated step in hormone-regulated steroidogenesis, the intramitochondrial transfer of cholesterol to the P450scc enzyme (for review, see [13]). Illustrating the essential role of StAR in steroid hormone synthesis, mutations in the StAR gene in humans cause lipoid congenital adrenal hyperplasia, a lethal condition that results from the inability of newborns to synthesize adequate levels of steroids [14]. Moreover, the phenotype of StAR knockout mice mirrors that of humans, as these mice are also unable to synthesize steroids to any great degree [15].
Because StAR mediates the intramitochondrial transfer of cholesterol to the P450scc enzyme, it seemed possible that lindane may reduce cholesterol accessibility to P450scc by blocking StAR protein expression. Therefore, the present study was performed to determine the effects of -, -, and -HCH on 1) steroid production; 2) total cellular protein synthesis; 3) P450scc and 3ß-hydroxysteroid dehydrogenase I (3ß-HSD) steroidogenic enzyme activity, protein, and mRNA levels; 4) StAR protein, mRNA levels, and gene transcription; and 5) protein kinase A (PKA) activity in mouse MA-10 Leydig tumor cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Waymouths MB 752/1 medium, horse serum, gentamicin sulfate, lyophilized trypsin-EDTA, PBS with Ca2+ and Mg2+ (PBS+), and PBS saline without Ca2+ and Mg2+ (PBS-) were purchased from Gibco Life Technologies (Gaithersburg, MD). [1,2,6,7-N-3 H(N)]-Progesterone (specific activity [SA], 97 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Antibodies to progesterone were obtained from Holly Hills Biological (Hillsboro, OR). Percoll and Dextran T70 were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Nunc cell culture dishes, charcoal (Norit), trichloroacetic acid, scintiverse BD, and sodium bicarbonate were obtained from Fisher Scientific (Houston, TX). Acrylamide, bis acrylamide, and SDS were purchased from Bio-Rad (Hercules, CA). -HCH was obtained from Aldrich (Milwaukee, WI). -HCH and -HCH, BSA, dibutyryl cAMP ([Bu]2cAMP), and 22(R)-hydroxycholesterol (22R-HC) were purchased from Sigma (St. Louis, MO). Rabbit antisera to amino acids 88–98 of mouse StAR protein were purchased from Research Genetics (Huntsville, AL). Rabbit antisera to amino acids 421–441 of rat P450scc enzyme were purchased from Chemicon (Temecula, CA). Antisera to purified mouse 3ß-HSD was a generous gift from Dr. A. Capponi, University of Geneva (Geneva, Switzerland). Mouse monoclonal antibody (20E8-C12) to bovine cytochrome oxidase subunit IV was obtained from Molecular Probes (Eugene, OR). Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G was purchased from Amersham (Arlington Heights, IL). StAR cDNA was previously cloned in our laboratory [16]. Bovine P450scc cDNA was a generous gift from Dr. M. Waterman, Vanderbilt University (Nashville, TN); mouse 3ß-HSD I cDNA was a generous gift from Dr. A. Payne, Stanford University (Stanford, CA); mouse L19 cDNA and 18S rRNA cDNA were generous gifts from Dr. G. Cornwall, Texas Tech University Health Sciences Center (Lubbock, TX).
MA-10 Cell Culture
The mouse MA-10 Leydig tumor cell line was a generous gift from Dr. Mario Ascoli, University of Iowa College of Medicine (Iowa City, IA). Cells were maintained in Waymouths MB 752/1 medium plus 15% horse serum at 37°C, 5% CO2 as described previously [17]. For dose-response, time-course, steroidogenic enzyme activity, and reversibility studies, 75 000 cells were seeded into each well of a 96-well plate and grown overnight. For nuclear run-on analysis, cells were grown to 80% confluency in 25 x 25 cm tissue culture dishes. For the remaining studies, 1.5 x 106 cells were plated into 100-mm culture dishes and grown until they were 80% confluent. For all experiments, medium was removed, cells were washed twice with PBS+, and serum-free Waymouths medium containing the appropriate treatment was placed onto the cells.
Treatment of Cells
Stimulation of MA-10 cells was performed using a maximal stimulatory dose of (Bu)2cAMP (1 mM). In some studies, optimal concentrations of 22R-HC (25 µM) was provided as a steroidogenic substrate. All treatments were performed in serum-free media. Final concentrations of dimethyl sulfoxide used as a chemical solvent were less than 0.4%, and were included in the control groups.
Dose-Response and Time-Course Studies
In dose-response and time-course studies, the effects of xenobiotics on steroidogenesis and total cellular protein synthesis were determined. MA-10 cells grown in 96-well plates were stimulated with (Bu)2cAMP in the presence or absence of various amounts of xenobiotics for 2 or 4 h. IC50 values were calculated as slope of the linear regression line obtained from Scatchard/Hofstee plots of steroidogenesis dose-response data.
Radioimmunoassay
Quantitation of progesterone in the medium was performed by radioimmunoassay (RIA) as previously described [18]. Standard curves were prepared in serum-free Waymouths medium. Analysis of RIA data was performed using a computer program specifically designed for this purpose. Data are expressed as ng/ml of media.
Determination of Total Cellular Protein Synthesis
To determine the effects of compounds on total protein synthesis, cells grown in 96-well plates were treated as described earlier with the inclusion of 5 µCi/ml Expre35S35S Protein Labeling Mix (SA 1000 Ci/mmol, New England Nuclear). A zero time point control was taken in which the Expre35S35S Protein Labeling Mix was added and immediately removed. Also, determination of total protein content by a modification of the method according to Bradford [19] was performed on identically plated cells that were not treated with Expre35S35S. Following treatment, media was removed and cells were solubilized for 2 h in 0.25 M NaOH at 37°C. Next, an equal volume of cold 20% trichloroacetic acid (TCA) was added and protein was precipitated overnight at 4°C. TCA-precipitable material was transferred onto glass fiber filters using a 1225 Sampling Manifold (Millipore, Bedford, MA) and rinsed with 5% TCA, dried, and counted in a liquid scintillation counter. Results were reported as counts per min/mg protein (2 or 4 h) minus counts per minute/mg protein sample (0 h).
Determination of P450scc and 3ß-HSD Activity and Reversibility
The effects of xenobiotics on the combined activities of the P450scc and 3ß-HSD enzymes were determined by adding 22R-HC to MA-10 cells in the presence or absence of the xenobiotic for 2 h and measuring progesterone production. To determine reversibility, cells were then rinsed with PBS+, allowed to recover for 24 h in serum containing medium, and incubated again for 2 h with (Bu)2cAMP and/or 22R-HC. Then, progesterone in the media was measured.
Isolation of Mitochondria and Western Blot Analysis
MA-10 cells grown in 100-mm dishes were stimulated with (Bu)2cAMP in the presence of or absence of HCH for 4 h. Mitochondria were isolated by homogenization and differential centrifugation [16]. Then, Western blot analysis of mitochondrial protein was performed as previously described [20]. Following detection of StAR, the membrane was stripped in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol at 70°C for 30 min, washed in 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl twice for 10 min, and then successively probed with P450scc, 3ß-HSD, and cytochrome oxidase antisera. The bands of interest were quantitated using a BioImage Visage 2000 (BioImage Corp., Ann Arbor, MI) imaging system. Values obtained were expressed as integrated optical density units, as previously described [21].
Isolation of RNA
Cells were treated as described in Western blot analysis. Then, total RNA was isolated using Trizol reagent (Gibco BRL, Grand Island, NY), according to the manufacturer's protocol. RNA was quantitated and resuspended in RNA sample buffer (0.1x borate buffer, 48% formamide, 6.4% formaldehyde, 5.3% glycerol, and 0.27% bromophenol blue).
Northern Blot Analysis
Northern blot analysis was performed as previously described [22]. Twenty micrograms of total RNA were loaded into each well. Labeling of cDNA probes for mouse StAR, P450scc, 3ß-HSD, and 18S rRNA was achieved by random priming (Prime-It II; Stratagene, La Jolla, CA) using [-32P]deoxycytidene triphosphate (SA, 3000 Ci/mmol; New England Nuclear), according to the manufacturer's protocol. After hybridization, the blots were washed twice in 2x SSC and 1% SDS at room temperature for 30 min, and once in 0.1x SSC and 0.1% SDS at 65°C for 30 min. Following Northern blot analysis with StAR cDNA, blots were stripped by washing twice in 0.1x SSC and 1% SDS at 65°C for 30 min, and then successively probed with P450scc, 3ß-HSD, and 18S rRNA cDNA. The bands of interest were quantitated and values obtained were expressed as described earlier.
Isolation of Nuclei
MA-10 cells grown in 25 x 25 cm tissue culture dishes were stimulated with Bu2cAMP in the presence and absence of the appropriate xenobiotic for 4 h. Isolation of nuclei and nuclear run-on analysis were performed as described by Greenburg and Bender in Current Protocols in Molecular Biology [23]. Following treatment, cells were harvested with a rubber policeman and centrifuged for 5 min at 500 x g at 4°C. The cell pellet was resuspended in ice-cold sucrose I buffer (0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5% [v/v] Nonidet P-40 [NP-40], 10 mM Tris-HCl pH 8.0), and homogenized with 5 strokes of a Dounce homogenizer. To verify that nuclei were free of cytoplasmic tags, nuclei were inspected with an Olympus IMT-2 inverted microscope (Dexter Instrument Co., San Antonio, TX). Then, the homogenate was layered onto a sucrose cushion consisting of sucrose buffer II (2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 10 mM Tris-HCl pH 8.0), and centrifuged for 45 min at 30 000 x g at 4°C. The supernatant was discarded and the pellet containing nuclei was resuspended in ice-cold glycerol storage buffer (40% [v/v] glycerol, 5 mM MgCl2, 0.1 mM EDTA, 50 mM Tris-HCl pH 8.3), frozen on dry ice, and stored in liquid nitrogen.
Nuclear Run-On Analysis
Nuclei were thawed at room temperature. An equal volume of 2x reaction buffer (5 mM MgCl2, 0.3 M KCl, 5 mM DTT, 10 mM Tris-HCl pH 8.0) containing 50 µCi/ml [-32P]uridine triphosphate (UTP) (SA 3000 Ci/mmol; New England Nuclear), and 0.5 mM cold ATP, GTP, and cytidine triphosphate (CTP) (Clontech, Palo Alto, CA) was added. Then, transcription complexes were elongated by incubating samples in a shaking water bath for 30 min at 30°C. Following incubation, the reaction mixture was digested successively with 40 µg/ml DNAse I for 5 min at 30°C and 160 µg/ml proteinase K for 30 min at 42°C. DNAse I and proteinase K were obtained from Sigma. RNA was extracted with 5:1 phenol-chloroform, pH 4.3 (Fisher Scientific), precipitated by the addition of 10% TCA, collected onto 0.45-µm Millipore HA nitrocellulose filters (Millipore) using a vacuum manifold, and rinsed free of unincorporated nucleotides with 5% TCA. RNA captured onto filters was treated with 25 µg/ml DNAse I for 30 min at 37°C, eluted from filters with elution buffer (1% SDS, 5 mM EDTA, 10 mM Tris-HCl) for 10 min at 65°C, and treated with 30 µg/ml proteinase K for 30 min at 37°C. The resultant mixture was extracted with 5:1 phenol-chloroform, pH 4.3, and subjected to a 10-min digestion on ice with 0.2 M NaOH prior to quenching the reaction with 0.29 M Hepes. RNA was precipitated by adding 1/10 (v/v) of 3 M sodium acetate and 2.5 vol of 100% EtOH, and centrifuged for 30 min at 10 000 x g at 4°C. The resultant RNA pellet was resuspended in water and an aliqout was counted using a liquid scintillation counter. Equal numbers of nuclei were used in the in vitro transcription assay, and equal counts of RNA were hybridized to target StAR, L19 and 18S cDNA inserts and linearized, empty pCMV-5 vector previously immobilized to nylon membranes (Hybond N+) using a Bio-Dot SF microfiltration apparatus (Bio-Rad) according to the manufacturer's protocol. Prehybridization and hybridization were performed under the same conditions described for Northern blot analysis. Radioactivity was detected using a Phosphorimager 445 SI (Molecular Dynamics, Sunnyvale, CA). Signals were quantitated using ImageQuant v4.1 software (Molecular Dynamics) in "Volume" mode, which integrates the intensity of each pixel within the defined area. Values were obtained as arbitrary units.
Protein Kinase A Activity Determination
Cells were treated as described for Western blot analysis. PKA activity was measured with the SignaTECT cAMP-Dependent Protein Kinase Assay System (Promega, Madison, WI). This assay measures the transfer of 32P to the biotinylated PKA peptide substrate, Kemptide (LRRASLG). Following treatment, cells were collected in extraction buffer (25 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM ß-mercaptoethanol, 1 µg/ml leupeptin, and 1 µg/ml aprotinin), homogenized as described earlier, and centrifuged at 13 000 x g for 10 min. The supernatant was used directly for both kinase assay and protein measurement according to the method of Bradford [19]. Approximately 3 µg of protein was incubated for 10 min at 30°C in 40 mM Tris-HCl pH 7.4, 20 mM MgCl, 0.1 mg/ml BSA, 0.1 mM biotinylated Kemptide, 0.1 mM ATP, and 0.02 µCi/µl [-32P]dATP (SA 3000 Ci/mmol; New England Nuclear). Reactions were stopped by the addition of 7.5 M guanidine-HCl. Ten µl of the reaction mix were spotted onto a streptavidin-coated membrane that specifically binds biotinylated Kemptide. Unincorporated [-32P]dATP and nonbiotinylated protein were then removed by extensive washing. The incorporation of 32P into biotinylated Kemptide bound to the membrane was determined by liquid scintillation counting. PKA activity was expressed as picomoles 32P incorporated per minute per mg protein.
Statistical Analysis
Statistically significant differences were determined by one-way ANOVA and Fisher-Protected Least Square Difference multiple comparison using the software program Statview SE + Graphics (Abacus Concepts, Inc., Berkeley, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-, -, and -HCH on Progesterone Production and Total Cellular Protein Synthesis
Initial studies were performed to determine the effects of -, -, and -HCH on steroidogenesis and total protein synthesis. As Figure 1 shows, these compounds decreased progesterone production in a dosage-dependent manner (IC50 in µM: -HCH = 19.7 ± 2.70, -HCH = 21.6 ± 2.49, -HCH = 23.1 ± 1.82) without affecting total protein synthesis, indicating that they did not cause acute cellular toxicity or a general inhibition in translation. In fact, 50 µM -, -, and -HCH significantly (P < 0.01) reduced steroidogenesis by 64%, 74%, and 81%, respectively, without affecting total protein synthesis. Therefore, we chose to use this dose of -, -, and -HCH in subsequent studies.
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As Table 1 shows, these compounds also disrupted steroidogenesis over time without inducing a parallel decrease in total protein synthesis. Whereas the effects of -, -, and -HCH were not statistically different from each other at 2 h, the level of steroidogenic inhibition induced by -HCH was significantly (P < 0.01) less than that induced by - and -HCH by 4 h. Whereas -HCH reduced progesterone production by 55% at 2 and 4 h, -HCH decreased steroid production by 60%, at both time points. In contrast, -HCH reduced steroidogenesis by 35% at 2 h and by only 19% at 4 h, indicating that steroidogenesis in -HCH-treated cells partially recovered by 4 h.
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Effects of -, -, and -HCH on P450scc and 3ß-HSD Enzyme Activity, Expression, and Steroidogenesis Following a 24-Hour Recovery
To determine if the inhibitory effects of -, -, or -HCH on (Bu)2cAMP-stimulated progesterone production might be due to inhibition of the activities of the P450scc and/or 3ß-HSD, 22R-HC was provided as substrate and cells were treated for 2 h with -, -, or -HCH (Fig. 2, A–C). The water soluble cholesterol analogue, 22R-HC, was used because it can readily diffuse to the P450scc enzyme located on the inner mitochondrial membrane, bypassing the need for StAR-mediated cholesterol transfer. In addition, to determine if the effects of drugs on steroidogenesis are reversible, cells were rinsed, allowed to recover for 24 h in serum containing medium, and treated again for 2 h with (Bu)2cAMP, 22R-HC, or both (Fig. 2, D–F). Although -, -, and -HCH significantly (P < 0.01) reduced (Bu)2cAMP-stimulated steroidogenesis by 55%, 74%, and 86%, respectively, (Bu)2cAMP-stimulated progesterone production in these cells returned to control levels following a 24-h recovery. Whereas 22R-HC alone dramatically increased progesterone production, these HCH isomers did not reduce steroidogenesis driven by 22R-HC after a 2-h treatment or following a 24-h recovery, indicating that they did not alter P450scc or 3ß-HSD enzyme activity.
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Previous studies have shown that levels of the steroidogenic enzymes (e.g., P450 17-hydroxylase/17,20 lyase) can be significantly reduced without the loss of steroidogenic capacity [24]. Therefore, to confirm that these compounds do not affect the expression of the P450scc and 3ß-HSD enzymes, Western and Northern blot analyses were performed. Although these compounds significantly (P < 0.001) blocked steroidogenesis by 53%, 87%, and 93%, respectively (Fig. 3), the level of steroidogenic inhibition induced by -HCH was significantly (P < 0.01) less than that induced by - and -HCH. Western blot analysis of mitochondrial protein confirmed that these compounds did not alter P450scc or 3ß-HSD enzyme levels (Figs. 4–5, upper panels). Moreover, Northern blot analysis revealed that these compounds did not affect P450scc mRNA levels (Fig. 4, lower panel). It is interesting that -HCH significantly (P < 0.05) reduced 3ß-HSD mRNA levels by 32%, further demonstrating that these isomers differentially affected components of the steroidogenic pathway (Fig. 5, lower panel).
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Effects of -, -, and -HCH on StAR Expression
Because StAR protein mediates the transfer of cholesterol to the inner mitochondrial membrane, an action that constitutes the rate-limiting step in steroidogenesis, the effects of -, -, and -HCH on the levels of StAR protein were also determined. Western blot analysis revealed that these compounds significantly (P < 0.05) reduced StAR protein levels by 48%, 89%, and 76%, respectively (Fig. 6, upper panel). The decrease in StAR protein levels induced by -HCH was significantly (P < 0.01) less than that induced by -HCH. It is important that these HCH isomers reduced StAR protein levels in parallel with steroidogenesis (compare Fig. 6, upper panel, with Fig. 3).
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To determine if these compounds reduced StAR protein levels by reducing StAR mRNA expression, Northern blot analysis was performed. StAR mRNA consists of the 1.6-, 2.7-, and 3.4-kilobase (kb) transcripts, which comprise 18%, 10%, and 72%, respectively, of total StAR mRNA (Fig. 6, lower panel). Northern blot analysis revealed that these compounds reduced total StAR mRNA levels similarly by 43%, 38%, and 55%, respectively. The -HCH-induced decrease in StAR mRNA levels was statistically significant at P < 0.05. For -HCH, a reduction in StAR mRNA levels may account for the decrease in StAR protein levels; however, for - and -HCH, a reduction in StAR mRNA levels could not entirely account for the decrease in StAR protein levels.
Although the importance of the three different StAR transcripts is unknown at this time, these compounds decreased levels of the most abundant 3.4-kb StAR transcript (Fig. 6, lower panel). Specifically, -, -, and -HCH decreased levels of the 3.4-kb StAR transcript by 37%, 49%, and 49%, respectively. The - and -HCH-induced decrease in levels of the 3.4-kb transcript was statistically significant at P < 0.05.
Because -HCH reduced StAR mRNA levels in proportion to StAR protein levels, nuclear run-on analysis was performed to determine if this isomer reduced the rate of StAR gene transcription. Nuclear run-on analysis measures the rate of transcription of a given gene, allowing for the comparison of the rate of transcription between cells treated with and without -HCH. As Figure 7 shows, stimulation with (Bu)2cAMP increased the rate of StAR gene transcription fivefold; however, -HCH decreased the rate of StAR transcription by 75%.
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Effects of -, -, and -HCH on Protein Kinase A Activity
A reduction in PKA activity may provide a simple explanation for the reduction in StAR expression and steroidogenesis. However, these compounds did not affect the ability of PKA present in cell lysates to phosphorylate the PKA-specific substrate, Kemptide, indicating that -, -, and -HCH inhibit StAR protein expression distal to PKA activation (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-, - and -HCH likely inhibited (Bu)2cAMP-stimulated steroid production in MA-10 cells by disrupting StAR protein expression. The observation that these isomers inhibited steroid production in MA-10 cells by limiting substrate availability is identical to the situation seen in Y-1 adrenal tumor and isolated Leydig and granulosa cells [2, 6, 12]. Also, the accumulation of cholesterol within steroidogenic cells observed in lindane-treated animals resembles that seen in StAR knockout mice, suggesting that lindane may block StAR expression following chronic exposure in vivo [2, 3, 25].
It is interesting that the HCH stereoisomers differentially affected StAR protein expression and steroidogenesis. This observation is comparable to previous reports that demonstrate that these isomers differentially affect neuronal activity. Whereas lindane (-HCH) elicits neuronal excitability, tremors, and convulsions in insects and mammals (an action that explains the insecticidal activity of HCH), the isomer induces only mild convulsions, and the isomer fails to elicit convulsions [26–29]. As the present study shows, for -HCH, a reduction in StAR mRNA levels may account for the decrease in StAR protein. However, for - and -HCH, a reduction in StAR mRNA levels could not account for the level of StAR protein inhibition, indicating that - and -HCH also disrupted StAR protein expression post-transcriptionally. Perhaps differences in their intracellular metabolism or the mechanism by which they inhibit StAR protein expression may contribute to their differential effects on StAR protein expression and, thus, steroidogenesis. Furthermore, nuclear run-on analysis showed that -HCH reduced StAR mRNA levels by decreasing the rate of StAR transcription. Because -HCH did not alter the transcription rate of the gene encoding the ribosomal protein L-19, another RNA polymerase II-transcribed gene, the data suggest that the effects of -HCH on transcription may be more specific to StAR. Although StAR protein is regulated at both the transcriptional and post-transcriptional levels, the proteins involved in hormone-stimulated StAR protein expression are poorly defined [30–33]. Therefore, even though the levels at which -, -, and -HCH blocks StAR expression have been identified, further studies will be necessary to determine how lindane blocks its expression at each site.
A general reduction in protein synthesis could have explained the decrease in steroid production because steroidogenesis requires de novo protein synthesis [34]. However, studies measuring 35S-methionine incorporation into total cellular protein indicate that HCH did not inhibit protein synthesis. Also, inhibition of PKA activity could have explained the inhibition of steroid production because both MA-10 and adrenal cells require protein phosphorylation to produce steroids [35, 36]. However, these compounds did not affect the ability of PKA to phosphorylate a PKA-specific substrate when measured in lindane-treated cells in vitro. However, because the phosphorylation status of StAR protein and other proteins that are crucial for steroidogenesis were not directly assessed, changes in phosphorylation status cannot be excluded.
Several observations led us to hypothesize that environmental toxicants block steroidogenesis via the disruption of StAR protein expression. First, in contrast to the steroidogenic enzymes, which have long half-lives and are chronically regulated, StAR protein is not an enzyme, is acutely regulated, and its active form is highly labile and must be continuously synthesized in order for steroidogenesis to occur. Second, StAR protein mediates the rate-limiting step in steroidogenesis and, thus, steroidogenesis is very sensitive to disruptions in StAR protein expression. Finally, recent studies have shown that the herbicide, Roundup (Monsanto, St. Louis, MO), the pesticide Dimethoate (BASF Co., Agricultural Products Group, Research Triangle Park, NC), and the antifungal drugs, econazole and miconazole, inhibit steroidogenesis prior to the P450scc enzyme, which also implicates StAR protein as a potential target for these compounds [37, 38].
Whereas -HCH significantly reduced 3ß-HSD mRNA levels, none of the isomers tested altered the levels or the activities of the P450scc and 3ß-HSD enzymes. Previously, investigators demonstrated that lindane did not reduce P450scc activity in Y-1 adrenal cells following acute exposure [12], but did reduce 3ß-HSD activity in isolated granulosa and Leydig cells following chronic exposure in vivo [2, 6]. Because both the P450scc and 3ß-HSD enzymes are chronically regulated and possess long half-lives, a more protracted exposure to HCH may reduce the levels of these enzymes and contribute to the decline in the cells' steroidogenic capacity.
Not only does lindane disrupt steroidogenesis, but it also alters the function of other endocrine and reproductive system components and, together, these alterations contribute to the pathogenesis of lindane-induced reproductive toxicity. First, lindane decreases LH secretion in sheep [39], rats [40], and mice [8]. Second, lindane decreases oocyte-directed folliculogenesis [41] and inhibits rat myometrial contractions [42] in vitro by disrupting gap junction formation. Third, lindane acts as a direct toxicant in mouse [43] and bovine [44] embryos in culture, possibly due to the production of toxic superoxide free radicals [45]. Finally, in female rodents, lindane alters sexual receptivity [46], disrupts ovarian cyclicity [40, 47], and reduces uterine weight [48]. Lindane can also block the response of estrogen-dependent tissues to estradiol [48–50] by competing with estradiol for binding to the estradiol receptor [51]. In effect, the ability of lindane to disrupt the endocrine and reproductive systems at multiple levels defeats compensatory mechanisms that exist to maintain homeostasis.
In conclusion, our results strongly suggest that lindane inhibited steroidogenesis by reducing StAR protein-mediated cholesterol transfer. This reduction in steroid production likely contributes to the pathogenesis of lindane-induced reproductive function in laboratory animals. While a cause-and-effect relationship between the presence of lindane in the natural habitats of humans or wildlife and the development of reproductive dysfunction via endocrine disruption remains to be established, the potential for lindane to disrupt reproductive function is real. Humans and animals may be directly exposed to high concentrations of lindane during its manufacture or use as well as to mixtures of pesticides at low concentrations that may interact to produce additive or synergistic effects. Furthermore, HCH is a highly lipophilic compound and, thus, can concentrate to a higher degree in adipose tissue [52–54], reproductive tissues such as testes [55] and ovaries [56, 57], and in breast milk [54]. Thus, these findings underscore the need for further studies to assess the affects of lindane and other environmental pollutants on wildlife and humans in their natural habitats.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH grant HD17481 to D.M.S. L.P.W was supported by NIH grant T32-HD07271 and a scholarship from the Lubbock Achievement Rewards for College Scientists chapter. 
2 Correspondence. FAX: 806 743 2990; doug.stocco{at}ttmc.ttuhsc.edu
Accepted: May 16, 2000.
Received: March 1, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-isomer in rats. Neuropharmacology 1981; 20:1017–1024[CrossRef][Medline]This article has been cited by other articles:
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  G. La Sala, D. Farini, and M. De Felici Proapoptotic Effects of Lindane on Mouse Primordial Germ Cells Toxicol. Sci., April 1, 2009; 108(2): 445 - 451. [Abstract] [Full Text] [PDF]
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 M. L. Biggs, M. D. Davis, D. L. Eaton, N. S. Weiss, D. B. Barr, D. R. Doody, S. Fish, L. L. Needham, C. Chen, and S. M. Schwartz Serum Organochlorine Pesticide Residues and Risk of Testicular Germ Cell Carcinoma: A Population-Based Case-Control Study Cancer Epidemiol. Biomarkers Prev., August 1, 2008; 17(8): 2012 - 2018. [Abstract] [Full Text] [PDF]
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 M. R. Milnes, T. A. Bryan, Y. Katsu, S. Kohno, B. C. Moore, T. Iguchi, and L. J. Guillette Jr. Increased Posthatching Mortality and Loss of Sexually Dimorphic Gene Expression in Alligators (Alligator mississippiensis) from a Contaminated Environment Biol Reprod, May 1, 2008; 78(5): 932 - 938. [Abstract] [Full Text] [PDF]
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 P. Murugesan, M. Balaganesh, K. Balasubramanian, and J. Arunakaran Effects of polychlorinated biphenyl (Aroclor 1254) on steroidogenesis and antioxidant system in cultured adult rat Leydig cells J. Endocrinol., February 1, 2007; 192(2): 325 - 338. [Abstract] [Full Text] [PDF]
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 G. Werle-Schneider, A. Wolfelschneider, M. C. von Brevern, J. Scheel, T. Storck, D. Muller, R. Glockner, H. Bartsch, and M. Bartelmann Gene Expression Profiles in Rat Liver Slices Exposed to Hepatocarcinogenic Enzyme Inducers, Peroxisome Proliferators, and 17{alpha}-Ethinylestradiol International Journal of Toxicology, September 1, 2006; 25(5): 379 - 395. [Abstract] [Full Text] [PDF]
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 N. Aluru and M. M. Vijayan Aryl Hydrocarbon Receptor Activation Impairs Cortisol Response to Stress in Rainbow Trout by Disrupting the Rate-Limiting Steps in Steroidogenesis Endocrinology, April 1, 2006; 147(4): 1895 - 1903. [Abstract] [Full Text] [PDF]
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N. Aluru, R. Renaud, J. F. Leatherland, and M. M. Vijayan Ah Receptor-Mediated Impairment of Interrenal Steroidogenesis Involves StAR Protein and P450scc Gene Attenuation in Rainbow Trout Toxicol. Sci., April 1, 2005; 84(2): 260 - 269. [Abstract] [Full Text] [PDF]
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F.-C. Ke, S.-H. Fang, M.-T. Lee, S.-Y. Sheu, S.-Y. Lai, Y. J. Chen, F.-L. Huang, P. S Wang, D. M Stocco, and J.-J. Hwang Lindane, a gap junction blocker, suppresses FSH and transforming growth factor {beta}1-induced connexin43 gap junction formation and steroidogenesis in rat granulosa cells J. Endocrinol., March 1, 2005; 184(3): 555 - 566. [Abstract] [Full Text] [PDF]
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 L. J. Guillette Jr. and T. M. Edwards Is Nitrate an Ecologically Relevant Endocrine Disruptor in Vertebrates? Integr. Comp. Biol., January 1, 2005; 45(1): 19 - 27. [Abstract] [Full Text] [PDF]
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 O. I. Kalantzi, R. Hewitt, K. J. Ford, L. Cooper, R. E. Alcock, G. O. Thomas, J. A. Morris, T. J. McMillan, K. C. Jones, and F. L. Martin Low dose induction of micronuclei by lindane Carcinogenesis, April 1, 2004; 25(4): 613 - 622. [Abstract] [Full Text] [PDF]
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V. D. Shultz, S. Phillips, M. Sar, P. M. D. Foster, and K. W. Gaido Altered Gene Profiles in Fetal Rat Testes after in Utero Exposure to Di(n-butyl) Phthalate Toxicol. Sci., December 1, 2001; 64(2): 233 - 242. [Abstract] [Full Text] [PDF]
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C. Mauduit, I. Goddard, V. Besset, E. Tabone, C. Rey, F. Gasnier, F. Dacheux, and M. Benahmed Leukemia Inhibitory Factor Antagonizes Gonadotropin Induced-Testosterone Synthesis in Cultured Porcine Leydig Cells: Sites of Action Endocrinology, June 1, 2001; 142(6): 2509 - 2520. [Abstract] [Full Text] [PDF]
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