Dose-Dependent Alterations in Gene Expression and Testosterone Synthesis in the Fetal Testes of Male Rats Exposed to Di (n-butyl) phthalate

Kim P. Lehmann, Suzanne Phillips, Madhabananda Sar, Paul M. D. Foster1 and Kevin W. Gaido2

CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709

Received March 19, 2004; accepted May 5, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to di (n-butyl) phthalate (DBP) in utero impairs the development of the male rat reproductive tract. The adverse effects are due in part to a coordinated decrease in expression of genes involved in cholesterol transport and steroidogenesis with a resultant reduction in testosterone production in the fetal testis. To determine the dose-response relationship for the effect of DBP on steroidogenesis in fetal rat testes, pregnant Sprague-Dawley rats received corn oil (vehicle control) or DBP (0.1, 1.0, 10, 50, 100, or 500 mg/kg/day) by gavage daily from gestation day (GD) 12 to 19. Testes were isolated on GD 19, and changes in gene and protein expression were quantified by RT-PCR and Western analysis. Fetal testicular testosterone concentration was determined by radioimmunoassay. DBP exposure resulted in significant dose-dependent reductions in mRNA and protein concentration of scavenger receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage, 3ß-hydroxysteroid dehydrogenase, and cytochrome P450c17. Testicular testosterone was reduced at doses of 50 mg/kg/day and above. Whole-testis expression of peripheral benzodiazepine receptor (PBR) mRNA, which functions with StAR to transport cholesterol across the mitochondrial membrane, was upregulated following exposure to DBP at 500 mg/kg/day. By immunocytochemistry, however, PBR protein was reduced in interstitial cells and also expressed but not reduced in gonocytes. Our results demonstrate a coordinate, dose-dependent reduction in the expression of key genes and proteins involved in cholesterol transport and steroidogenesis and a corresponding reduction in testosterone in fetal testes following maternal exposure to DBP, at dose levels below which adverse effects are detected in the developing male reproductive tract. Alterations in gene and protein expression and testosterone synthesis may serve as sensitive indicators of testicular response to DBP.

Key Words: di (n-butyl) phthalate; in utero exposure; male reproductive development; antiandrogen; molecular mechanisms; androgen receptor; dose response; steroidogenesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Di (n-butyl) phthalate (DBP) is used as a plasticizer and as a stabilizer in numerous consumer products, including hair sprays, cosmetics, latex adhesives, inks, and caulking. Because of its widespread use, there is potential for human exposure in the general population on a daily basis. Estimates for human exposure to DBP range from 0.84 to 113 µg/kg/day (Blount et al., 2000Go; Kohn et al., 2000Go). DBP is rapidly metabolized and eliminated and does not bioaccumulate (Saillenfait et al., 1998Go; Tanaka et al., 1978Go).

Maternal doses of DBP that are without apparent effects in the dam (100–500 mg/kg/day) can adversely affect development of the male rat reproductive tract. Adverse effects include absent or deformed epididymides, cryptorchidism, hypospadias, reduced fertility, and Leydig cell adenoma (Mylchreest et al., 1998Go, 1999Go, 2000Go). The effects of DBP on the developing male reproductive tract are similar, although not identical, to the effects of antiandrogens such as flutamide and linuron (McIntyre et al., 2000Go, 2001Go, 2002Go). Unlike flutamide and linuron, however, neither DBP nor its primary metabolite monobutyl phthalate (MBP) interacts with the androgen receptor (Foster et al., 2001Go). The antiandrogenic effects of DBP are due instead to decreased testosterone synthesis as a result of a reduction in expression of genes involved in cholesterol transport and testosterone synthesis (Barlow et al., 2003Go; Shultz et al., 2001Go).

In a previous DBP dose-response study (using doses of 0.5, 50, 100, and 500 mg/kg/day), male rats exposed in utero to 100 and 500 mg DBP/kg/day showed a dose-dependent increase in retained nipples, an indicator of reduced androgen status during development (Mylchreest et al., 2000Go). Other adverse effects such as hypospadias, absent or deformed epididymides, vas deferens, seminal vesicles, and ventral prostate were observed only at the highest dose level. No statistically significant adverse effects were observed in the offspring of dams treated with ≤50 mg DBP/kg/day. We repeated the dose-response study to examine the dose-response relationship for the effect of DBP on gene and protein expression and testosterone concentration in the fetal testes. A broader range of dose levels was selected to incorporate a dose level (0.1 mg/kg/day) approximately equivalent to the maximum estimated level of exposure for the general population in the United States (Blount et al., 2000Go; Kohn et al., 2000Go). Fetal testes were examined on GD 19 based on our previous studies that showed significant reductions in the expression of genes involved in cholesterol transport and testosterone synthesis at this time (from GD 12–19) following DBP treatment (Barlow et al., 2003Go; Shultz et al., 2001Go). We demonstrate a coordinate dose-dependent decrease in fetal testicular testosterone concentration and expression of genes and their corresponding proteins involved in cholesterol transport and testosterone synthesis in the fetal testis at dose levels below the levels at which adverse effects are detected.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal model. Sprague-Dawley outbred rats were time-mated at Charles River Laboratories, Inc. (Raleigh, NC) and shipped to the CIIT Centers for Health Research on GD 0, the day sperm was detected in the vaginal smear. Dams were assigned to a treatment group by body weight randomization using Provantis (Instem LSS, Stone, UK) with seven animals in the control group and five animals in each of the DBP-dosed groups. Animals were housed in the CIIT animal facility, accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, in a humidity- and temperature-controlled, HEPA-filtered, mass air–displacement room. The room was maintained on a 12-h light/dark cycle at approximately 22 ± 4°C with a relative humidity of approximately 30–70%. Animals were identified by ear tags and cage cards and housed individually in polycarbonate cages with Alpha-dri cellulose bedding (Shepherd Specialty Papers, Kalamazoo, MI). Rodent diet NIH-07 (Zeigler Brothers, Gardener, PA) and reverse-osmosis water were provided ad libitum. This study followed Federal guidelines for the care and use of laboratory animals and was approved by the Institutional Animal Care and Use Committee at CIIT.

Study design. Dams were treated by gavage (1 ml/kg) daily from GD 12–19 with corn oil vehicle (Sigma Chemical Co., St. Louis, MO) or DBP (Aldrich Chemical Co., Milwaukee, WI) in corn oil at 0.1, 1, 10, 50, 100, or 500 mg/kg/day. Purity and concentration of all doses were verified using a Hewlett Packard 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA). This study was repeated a second time with a 30-mg/kg/day dose group, in addition to the above-mentioned dose level groups, to generate samples for the testosterone radioimmunoassay (RIA). The highest dose level was chosen based on our previous studies showing that 500 mg/kg/day produced significant changes in gene expression in the male offspring without maternal toxicity or fetal death (Barlow and Foster, 2003Go; Shultz et al., 2001Go). The lowest dose level was selected based on current estimates for human exposure, which reach as high as 0.113 mg/kg/day (Blount et al., 2000Go; Kohn et al., 2000Go).

Dam body weights were recorded on GD 4 and daily during the dosing period. All dams were euthanized on GD 19 by carbon dioxide asphyxiation. Fetuses were removed by cesarean section and body weights were recorded. All fetuses were euthanized by decapitation and then sexed by internal examination of the reproductive organs. The right and left testes and epididymides were removed from male fetuses and separated using a dissecting microscope with transillumination. Testes were snap-frozen in liquid nitrogen in separate vials and stored at –80°C.

Real-time quantitative RT-PCR. Total RNA was isolated from the testes of five individual fetuses representing four to five litters per treatment group using RNA STAT-60 reagent (Tel-Test, Friendswood, TX). Subsequent reverse transcription (RT) reactions, quality control for RT reactions, and quantitative PCR reactions were performed as described previously (Barlow et al., 2003Go). Rat-specific primers and probes were designed for the genes of interest (Tables 1 and 2) using Primer Express software (Applied Biosystems, Foster City, CA) with the following parameters: low Tm = 60°C; high Tm = 64°C; optimum Tm = 62°C; amplicon length = 70 to 150 base pairs; primer length = 12 to 25 base pairs; and optimum length = 20 base pairs.


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TABLE 1 Primer Sets for Real-Time Quantitative RT-PCR Analyses

 

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TABLE 2 Probe Sequences for Real-Time Quantitative RT-PCR Analyses

 
Western blot analysis. Whole testes from four individual fetuses per treatment group were solubilized in Laemmli buffer, and protein concentration was determined by using a bicinchoninic acid (BCA) protein assay kit and following the manufacturer's protocol (Sigma Chemical Co.). Samples were heated at 95°C for 5 min, and then equal concentrations of protein were added to each lane on a 12% SDS–PAGE minigel (Bio-Rad Laboratories, Hercules, CA). Proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were incubated for 1 h at room temperature in blocking Tris-buffered saline with 0.1% Tween-20 (TBST) buffer containing 5% nonfat milk. Immunoblot analyses of membranes were performed by incubating with the following primary antibodies at 4°C overnight, with the exception of SR-B1 incubated at room temperature for 1 h: StAR (rabbit polyclonal IgG, ABR, Golden, CO); P450scc (rabbit polyclonal, US Biological, Swampscott, MA); SR-B1 (rabbit polyclonal, Novus Biologicals, Littleton, CO); and CYP17 (rabbit polyclonal, provided by Dr. Dale Buchanan Hales, University of Illinois at Chicago). Membranes were washed in TBST for 45 min followed by incubation with secondary antibody donkey antirabbit IgG conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ). Positive bands were detected by chemiluminescence with the ECL Plus Western Blot Detection kit (Amersham Biosciences) after a final 1 h wash in TBST. Densitometry was performed using FluorChem version 2.0 analysis software (Alpha Innotech Corporation, San Leandro, CA).

Immunohistochemistry. GD 19 fetal testis from control rats and rats treated with 500 mg DBP from the present study (as well as from a previous study, Barlow et al., 2003Go) were immersion-fixed in 10% neutral buffered formalin for 24 h and then transferred to 70% ethanol. Next, the tissues were embedded in paraffin, sectioned at 5 µm, placed on charged slides, and stored at room temperature until processed. At processing, sections were deparaffinized, treated with 3% H2O2 in water for 10 min to block endogenous peroxidase activity, and heated in a microwave for 3 min in citrate buffer (1:10 dilution in deionized water, pH 5.5–5.7; BioGenex, San Ramon, CA) for antigen retrieval. The sections were treated with 10% powdered nonfat milk for 20 min following 2% normal goat serum in PBS for 10 min to reduce nonspecific staining. The sections were then incubated with the primary antibodies PBR (rabbit polyclonal IgG, 2 µg/ml) and Insl3 (rabbit polyclonal IgG, 5 µg/ml) overnight at 4°C. Rabbit anti-PBR was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit anti-Insl3 was obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA). Following incubation with the primary antibodies, the slides were washed in PBS for 5 min followed by incubation with a biotinylated secondary antibody, antirabbit IgG (1:100), and then with avidin-biotin peroxidase (Vector Labs, Burlingame, CA) for 30 min at room temperature (Sar and Welsch, 1999Go). The sections were treated with liquid diamino benzidine (BioGenex) for 3 min, washed in water, counterstained with hematoxylin, and mounted with Paramount. Antibody specificity was confirmed by excluding incubation with the secondary antibody.

Radioimmunoassay. Fetal testicular testosterone steroid hormone concentration was determined from three to four individual fetuses from one to four litters per dose group, following the previously described method (Shultz et al., 2001Go) using the Testosterone CT kit (ICN Pharmaceuticals, Costa Mesa, CA). Testes were homogenized in 100 µl of PBS-Gel buffer; the homogenate was then extracted three times with a total of 1 ml of a fresh mixture of ethylacetate and chloroform (4:1). Extracts were dried under nitrogen and resuspended in 1 ml methanol. An aliquot (5 or 10 µl) was taken for analysis. An equal volume of extraction solvent was added to standards (0.25–128 pg of steroid hormone per tube) and recovery tubes (25 µl 3[H] steroid hormone, 5000 dpm) and dried under nitrogen. Dextran-coated charcoal (DCC) stripped serum (25 µl) was added to recovery tubes. Rabbit antitestosterone hormone antibody (ICN) was diluted (1:800,000) with phosphate-buffered saline containing 0.01% {gamma}-globulin and 0.1% gelatin (PBS-Gel); 100 µl was added to each tube, gently mixed, and incubated overnight at 4°C. 125I-testosterone hormone (100 µl, 15,000 cpm) was added, and tubes were incubated for 4 h at room temperature. The second antibody (100 µl; goat antirabbit IgG diluted 1:9–1:11, ICN) was added, and tubes were incubated for 1 h in a water bath at 38°C. Following the addition of PBS-Gel (3 ml), tubes were centrifuged for 1 h at 1500 x g. The supernatant was decanted, the tubes blotted on absorbent paper, and the pellet counted for 2 min per tube in a Cobra gamma counter (D5005, Packard Instrument Co., Downers Grove, IL).

Oil red O histochemistry. Frozen sections from GD 19 testis from four to five separate rat fetuses from different dams per treatment group, except for the control group that had 10 individual fetuses from 6 dams, were cut and placed on slides. Oil red O staining was performed as previously described (Pearse, 1996Go), with the exception that hematoxylin was not used on sections for lipid quantitation. Image-Pro Plus software (version 4.5; Media Cybernetics, Carlsbad, CA) was used to quantify the total area of the section and the area of oil red O stain to give the relative amount of lipid per section.

Statistical analyses. All statistical analyses were conducted using either JMP version 5.0.1 or SAS software (SAS Institute, Cary, NC). In all analyses, the litter was the experimental unit. Gene expression data were analyzed by Dunnett's test comparing the relative expression ratios from each nonzero dose group to the control. The error term for the Dunnett's test was generated by a one-way ANOVA. Relative expression ratios were calculated as described previously (Barlow et al., 2003Go) using the equation set forth (Pfaffl, 2001Go) in which efficiencies for both the gene of interest and the calibrator GAPDH were used. Analyses of relative expression ratios were considered to be statistically significant for p < 0.05.

Radioimmunoassay data were analyzed by Dunnett's test comparing log10-transformed testosterone concentrations from each nonzero dose group to the control. The error term for the Dunnett's test was generated by a two-way ANOVA. The two factors used in this analysis were dose and extract preparation day. A trend analysis was also performed after fitting the data to a one-way ANOVA model using S-Plus. Analyses of testosterone concentrations were considered to be statistically significant for p < 0.05. Western blot data were analyzed by Dunnett's test comparing raw intensity values from each nonzero dose group to the control. The error term for the Dunnett's test was generated by a two-way ANOVA. The two factors used in this analysis were dose and membrane. Analyses of protein expression values were considered to be statistically significant for p < 0.05. Lipid data were analyzed by Dunnett's test comparing the area of stain per section from each nonzero dose group to the control. The error term for the Dunnett's test was generated by a one-way ANOVA with subsampling, which indicated that the subsampling and experimental errors could be combined. Analyses of oil red O values were considered to be statistically significant for p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pregnant rats received either corn oil (vehicle control) or DBP (0.1, 1.0, 10, 50, 100, or 500 mg/kg/day) by gavage daily from GD 12 to 19. DBP exposure resulted in a dose-dependent decline in expression of genes involved in cholesterol transport and steroidogenesis (Fig. 1). The dose-response curves for scavenger receptor class B type 1 (SR-B1), steroid acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) were similar, with a reduction in expression of approximately 50% for each gene at 50 mg/kg/day and 80% at 500 mg/kg/day. SR-B1 mRNA was also significantly reduced at 1.0 mg/kg/day. 3ß Hydroxysteroid dehydrogenase (3ß-HSD) gene expression showed a significant reduction at 0.1 and 1.0 and a further reduction at doses of 50 mg/kg/day and above. Cytochrome P450c17 (CYP17) was significantly reduced only at 500 mg/kg/day. Expression of peripheral benzodiazepine receptor (PBR) mRNA was increased in response to DBP (Fig. 1F). PBR is a mitochondrial-membrane-spanning receptor and, like StAR, is essential for cholesterol transport across the mitochondrial membrane (Papadopoulos et al., 1997Go). Sterol regulatory element–binding protein (SREBP) gene expression was not altered following DBP exposure (data not shown).



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FIG. 1. Real-time quantitative RT-PCR analyses of testicular mRNA collected on gestation day (GD) 19 from control and DBP-exposed fetuses. Gene expression values from DBP-exposed testes are expressed relative to control values and represent the average ± SEM from five separate rat fetuses from different dams per treatment group. (A) Scavenger receptor B-1 (SR-B1); (B) steroid acute regulatory protein (StAR); (C) cytochrome P450 side-chain cleavage (P450scc); (D) CYP17; (E) 3ß-hydroxysteroid dehydrogenase (3ß-HSD); (F) peripheral benzodiazepine receptor (PBR); (G) testosterone-repressed prostate message-2 (TRPM-2); (H) c-Kit; and (I) insulin-like factor 3 (Insl3). *p < 0.05.

 
Previously, we showed that in utero exposure to DBP also resulted in the altered expression of genes involved in cell survival, including c-Kit (also known as stem cell factor receptor) and testosterone-repressed prostate message 2 (TRPM-2; Barlow et al., 2003Go; Shultz et al., 2001Go). We examined the dose-response relationship for the effect of DBP on the expression of these two genes. Similar to 3ß-HSD, expression of c-Kit (Fig. 1H) was significantly reduced at 0.1 and 1.0 and further reduced at ≥50 mg/kg/day. In contrast, TRPM-2 (Fig. 1G) was induced only at the highest dose level of DBP.

Protein expression, as determined by Western analysis, mirrored the changes in gene expression with significant reductions in SR-B1 and StAR occurring at doses ≥50 mg/kg/day (Fig. 2). P450scc protein was significantly reduced only at 500 mg/kg/day (Fig. 2C), whereas the mRNA for P450scc was significantly reduced at doses ≥50 mg/kg/day (Fig. 1C).



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FIG. 2. Western analyses of testicular protein collected on GD 19 from control and DBP-exposed fetuses. (A) Representative immunoblots for SR-B1, P450scc, StAR, and CYP17. (B) Average relative protein expression levels ± SEM from four separate rat fetuses from different dams per treatment group. *P<0.05

 
Accurate quantification by Western analysis of 3ß-HSD and PBR could not be obtained. PBR was examined further by immunocytochemistry. Staining for PBR was reduced in the interstitial cells. PBR expression was similarly expressed in the gonocytes of control and DBP-exposed testes (Figs. 3A and 3B). The reduction of PBR in the testicular interstitial cells correlates well with the reduction of StAR and other proteins involved in cholesterol transport and steroidogenesis. Expression of PBR in gonocytes has not been previously reported, and the relevance of this observation remains to be determined. In utero exposure to DBP doses of 250 and 500 mg/kg/day caused intra-abdominal cryptorchidism (Barlow and Foster, 2003Go; Mylchreest et al., 1998Go, 2000Go). The pattern of DBP-induced cryptorchidism is similar to the cryptorchidism that occurs with the insulin-like growth factor 3 (Insl3) knockout mouse (Emmen et al., 2000Go). In contrast, androgen receptor antagonists such as flutamide can cause inguinal cryptorchidism (Mylchreest et al., 1999Go). We examined the dose-response relationship for DBP on Insl3 to determine whether the cryptorchidism induced by DBP was due to altered expression of this gene. Insl3 mRNA was significantly reduced at 500 mg/kg/day DBP (Fig. 1I). By immunohistochemistry, we showed reduced staining for Insl3 in interstitial cells from DBP-exposed fetal testes (Figs. 3C and 3D).



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FIG. 3. Immunohistochemical staining of GD 19 testis from (A and C) control males and (B and D) DBP-exposed males (500 mg/kg/day) for (A and B) PBR and (C and D) Insl3. In control testis, interstitial cells (IC) showed strong immunostaining for PBR (A) while DBP-exposed testis (B) had little or no immunostaining of interstitial cells. In control testis (C), interstitial cells showed increased staining for Insl3 compared to the DBP-exposed (D) testis. G, gonocytes; magnification x280.

 
Intratesticular testosterone was significantly reduced at doses ≥50 mg/kg/day, as determined by radioimmunoassay (Fig. 4). We reported previously that high-dose exposure to DBP resulted in a reduction in Leydig cell lipid content, as determined by oil red O staining (Barlow et al., 2003Go). We examined the dose response for this effect and found a significant reduction only at the highest dose (Fig. 5).



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FIG. 4. Fetal testicular testosterone concentration of fetal testes collected on GD 19 from control and DBP-exposed fetuses. Values are expressed relative to control values and represent the average ± SEM from three to four separate rat fetuses from one to four dams per treatment group. *p < 0.05.

 


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FIG. 5. The effect of DBP on fetal testicular lipid. Fetal testes from control and DBP-treated male rat fetuses were collected on GD 19 and stained using oil red O. (A) Control testes; (B) representative image of testes from 500 mg DBP/kg/day treatment group. (C) Image-Pro Plus software was used to quantify the total area of the section and the area of oil red O stain to give the relative amount of lipid per section. Values are expressed relative to control values and represent the average ± SEM from four to five separate rat fetuses from different dams per treatment group, except for the control group that had 10 individual fetuses from six dams. *p < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we demonstrate the dose-response relationship for an effect of DBP on key steps in the steroidogenic pathway and correlate changes in gene and protein expression with a corresponding dose-dependent reduction in fetal testis testosterone concentration. Fetal testicular testosterone was significantly reduced at DBP doses ≥50 mg/kg/day. In an earlier study that examined 20 litters per dose group, we established 50 mg DBP/kg/day as the no observable adverse effect level (NOAEL) for effects on male reproductive tract development (Mylchreest et al., 2000Go). Thus, while the reduction in fetal testicular testosterone production is not overtly adverse at 50 mg DBP/kg/day, our current results indicate that some reduction in steroidogenic gene and protein expression and fetal testicular testosterone can occur at dose levels below those at which significant effects on reproductive tract development can be detected. Our results establish 50 mg DBP/kg/day as a lowest observable effect level (LOEL) and 10 mg DBP/kg/day as a no observable effect level (NOEL) for reductions in genes and proteins associated with testosterone production together with reductions in intratesticular testosterone.

The steroidogenic enzymes responsible for the conversion of cholesterol to testosterone include P450scc, 3ß-HSD, CYP17, and 17ß-HSD. We demonstrated previously that P450scc, 3ß-HSD, and CYP17, but not 17ß-HSD, are reduced in DBP-exposed fetal testes (Barlow et al., 2003Go; Shultz et al., 2001Go). Of the steroidogenic enzymes, 3ß-HSD was the most sensitive to respond following DBP treatment with a significant decrease in mRNA at 0.1 and 1.0 mg/kg/day. Fetal testicular testosterone was not altered at these doses and the biological relevance of this reduction in 3ß-HSD mRNA at low-dose levels remains to be determined.

Cholesterol uptake into the cell is mediated by SR-B1, also known as high-density lipoprotein receptor (Acton et al., 1996Go). Intracellular cholesterol is transported to the outer mitochondrial membrane, where StAR mediates the transfer of cholesterol from the outer to the inner mitochondrial membrane (Stocco, 2001Go). SR-B1 mRNA was significantly reduced at 1 mg/kg/day and further reduced at doses ≥50 mg/kg/day. SR-B1 protein was also reduced by 20 ± 10% at 1 mg/kg/day, although this reduction was not statistically significant. SR-B1 protein was significantly reduced at ≥50 mg/kg/day.

StAR works in concert with PBR to regulate cholesterol transport across the mitochondrial membrane (West et al., 2001Go). While total testicular PBR mRNA was increased following DBP exposure, PBR protein was reduced in the interstitial cells of the fetal testes following DBP exposure. The reason for this apparent discrepancy between total testicular PBR mRNA and interstitial cell PBR protein levels is not known but may indicate enhanced PBR protein turnover or another post-transcriptional event. Alternatively, this difference may be due to differential regulation of PBR in fetal testicular gonocytes and Leydig cells. PBR is primarily expressed in testicular Leydig cells under normal conditions, and our results showing a decrease in PBR protein in interstitial cells following phthalate treatment are in agreement with a previously published study (Gazouli et al., 2002Go). In that study, treatment of 12-week-old mice with 1 g/kg/day di-2-ethylhexyl phthalate (DEHP) caused a reduction in PBR expression (Gazouli et al., 2002Go). Similarly, treatment of MA-10 mouse Leydig tumor cells in culture with mono (2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, also reduced PBR expression (Gazouli et al., 2002Go). Expression of PBR in fetal gonocytes has not been previously reported. However, low levels of radiolabeled PBR ligand binding in the rat seminiferous tubules has been shown, suggesting the presence of PBR in the Sertoli and germ cell population (De Souza et al., 1985Go).

Steroidogenically active Leydig cells synthesize and store fatty acids and cholesterol to help maintain steroidogenesis. Androgens upregulate this process through a cascade of events involving androgen-dependent activation of SREBP (Brown and Goldstein, 1998Go; Swinnen et al., 1997Go, 1998Go). Both DBP and flutamide, an androgen receptor–competitive antagonist, downregulate expression of genes involved in fatty acid and cholesterol synthesis, including long-chain-specific acyl-CoA, acetyl-CoA carboxylase, steryl sulfatase, and low-density lipoprotein receptor (Shultz et al., 2001Go). Downregulation of genes involved in cholesterol synthesis, together with the reduction of cholesterol import through downregulation of SR-B1, is likely the reason for the dose-dependent decrease in Leydig cell lipid content, as determined by oil red O staining.

During reproductive development, the fetal testes descend from a pararenal position through the abdominal wall and into the scrotal sac. Insl3, also known as relaxin-like factor, is produced by Leydig cells and is essential for gubernacular development and testicular descent from the pararenal through the abdomen (Nef and Parada, 1999Go; Zimmermann et al., 1999Go). Male mice deficient in Insl3 have bilateral intra-abdominal testes (Nef and Parada, 1999Go; Zimmermann et al., 1999Go). This form of cryptorchidism is similar to that which occurs following in utero exposure to DBP doses of 250 and 500 mg/kg/day (Barlow and Foster, 2003Go; Mylchreest et al., 1998Go). We have shown that Insl3 expression is suppressed following exposure to DBP doses >100 mg/kg/day, and that the gubernaculum is underdeveloped in male rats exposed gestationally to 500 mg/kg/day DBP (Barlow and Foster, 2003Go). Our results are in keeping with a recently published report of reduced Insl3 expression following fetal exposure to several different phthalates, including DBP (Wilson et al., 2004Go). Together, these studies suggest that phthalate-induced cryptorchidism is due to decreased Insl3 production by the fetal Leydig cell.

Fetal testes of rats exposed in utero to DBP contain focal regions of Leydig cell hyperplasia (Barlow and Foster, 2003Go; Mylchreest et al., 1999Go, 2000Go). Hyperplasia is not observed with DBP doses ≤100 mg/kg/day (Mylchreest et al., 2000Go). Fetal Leydig cell hyperplasia may be due in part to enhanced cell survival since these regions contain enhanced expression of two factors associated with cell survival, TRPM-2 and Bcl-2 (Shultz et al., 2001Go). We showed that the induction of TRPM-2 occurred at doses above 100 mg/kg/day, which correlates well with the appearance of the focal lesions.

C-Kit mRNA was significantly reduced at 0.1 and 1.0 mg DBP/kg/day and further reduced at DBP doses ≥50 mg/kg/day. Kit-ligand (Kitl), or stem cell factor, is produced as both a membrane-bound form and a secreted form by the Sertoli cell and is essential for normal gonocyte proliferation and survival. We demonstrated previously that Kitl is reduced in fetal Leydig cells following DBP exposure (Barlow et al., 2003Go). Mutation or knockout of either Kitl or its receptor (c-Kit) results in infertility due to germ cell loss (Feng et al., 1999Go; Mauduit et al., 1999Go; Ohta et al., 2000Go). Kitl has also been shown to influence Leydig cell steroidogenesis (Rothschild et al., 2003Go), and the effect of DBP on testosterone synthesis may be due, at least in part, to reduced stem cell factor signaling.

For several of the genes examined in this study (SR-B1, 3ß-HSD, and c-Kit), we found significant reductions in mRNA levels at DBP doses that approach maximal human exposure levels. The biological relevance of these alterations in gene expression at low dose levels remains to be determined, since no statistically significant observable adverse effects on male reproductive tract development have been identified at DBP doses <100 mg/kg/day, following the protocol we used in this study (Mylchreest et al., 2000Go), and since fetal testicular testosterone is reduced only at dose levels ≥50 mg/kg/day. The mRNA and corresponding proteins of these genes may be produced in excess and small changes in their expression levels may not significantly affect steroidogenesis. StAR transport of cholesterol across the mitochondrial membrane is generally considered the rate-limiting step in steroidogenesis (Stocco, 2001Go), and expression of StAR mRNA and protein is not significantly altered below 50 mg/kg/day. Our results indicate that alterations in the expression of SR-B1, c-Kit, and 3ß-HSD may be sensitive indicators of DBP exposure but not necessarily of adverse consequences to DBP.

Significance was not achieved at the 10 mg/kg/day dose for the genes that had significantly altered expression at 0.1 and 1.0 mg/kg/day (SR-B1, 3ß-HSD, and c-Kit). The values obtained at the 10 mg/kg/day dose were within the expected range of variability. Studies incorporating more litters and additional doses may be necessary to more accurately define the shape of the dose-response curve for these genes in the dose range of 1–50 mg/kg/day.

In summary, we report that gestational exposure to 50 mg DBP/kg/day results in the coordinate reduction of genes and their corresponding proteins involved in cholesterol transport and steroidogenesis, along with a reduction in intratesticular testosterone. This reduction in cholesterol transport proteins, steroidogenic enzymes, and intratesticular testosterone occurred at a dose at which no observable adverse effects on the developing male reproductive tract were detected. Our results indicate that alterations in gene and protein expression and testosterone synthesis are sensitive indicators of testicular response to DBP.


    ACKNOWLEDGMENTS
 
We thank Drs. Kamin Johnson, Li You, and Michael Shelby for their critical review of this report; Drs. Dennis House and Kejun Liu for their assistance with the statistical analyses of the data; and Dr. Barbara Kuyper for her editorial review. This study was supported by the National Institutes of Health Grant R21 ES011754-01.


    NOTES
 
1 Present address: National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709. Back

2 To whom correspondence should be addressed at CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, NC 27709. Fax: (919) 558-1300. E-mail: gaido{at}ciit.org.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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