CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709
Received March 19, 2004; accepted May 5, 2004
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ABSTRACT |
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Key Words: di (n-butyl) phthalate; in utero exposure; male reproductive development; antiandrogen; molecular mechanisms; androgen receptor; dose response; steroidogenesis.
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INTRODUCTION |
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Maternal doses of DBP that are without apparent effects in the dam (100500 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., 1998, 1999
, 2000
). 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., 2000
, 2001
, 2002
). Unlike flutamide and linuron, however, neither DBP nor its primary metabolite monobutyl phthalate (MBP) interacts with the androgen receptor (Foster et al., 2001
). 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., 2003
; Shultz et al., 2001
).
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., 2000). 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., 2000
; Kohn et al., 2000
). 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 1219) following DBP treatment (Barlow et al., 2003
; Shultz et al., 2001
). 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.
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MATERIALS AND METHODS |
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Study design. Dams were treated by gavage (1 ml/kg) daily from GD 1219 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, 2003; Shultz et al., 2001
). 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., 2000
; Kohn et al., 2000
).
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., 2003). 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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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., 2003) 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 5.55.7; 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, 1999
). 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., 2001) 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 (0.25128 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%
-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 1:91:11, 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, 1996), 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., 2003) using the equation set forth (Pfaffl, 2001
) 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.
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RESULTS |
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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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DISCUSSION |
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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., 2003; Shultz et al., 2001
). 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., 1996). 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, 2001
). 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., 2001). 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., 2002
). 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., 2002
). 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., 2002
). 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., 1985
).
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, 1998; Swinnen et al., 1997
, 1998
). Both DBP and flutamide, an androgen receptorcompetitive 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., 2001
). 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, 1999; Zimmermann et al., 1999
). Male mice deficient in Insl3 have bilateral intra-abdominal testes (Nef and Parada, 1999
; Zimmermann et al., 1999
). 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, 2003
; Mylchreest et al., 1998
). 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, 2003
). 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., 2004
). 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, 2003; Mylchreest et al., 1999
, 2000
). Hyperplasia is not observed with DBP doses
100 mg/kg/day (Mylchreest et al., 2000
). 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., 2001
). 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., 2003
). Mutation or knockout of either Kitl or its receptor (c-Kit) results in infertility due to germ cell loss (Feng et al., 1999
; Mauduit et al., 1999
; Ohta et al., 2000
). Kitl has also been shown to influence Leydig cell steroidogenesis (Rothschild et al., 2003
), 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., 2000), 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, 2001
), 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 150 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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.
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