ToxSci Advance Access originally published online on March 15, 2007
Toxicological Sciences 2007 97(2):512-519; doi:10.1093/toxsci/kfm055
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Published by Oxford University Press 2007.
Sensitivity of Fetal Rat Testicular Steroidogenesis to Maternal Prochloraz Exposure and the Underlying Mechanism of Inhibition
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* Department of Environmental and Molecular Toxicology, NC State University, Raleigh, North Carolina 27695
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, Research Triangle Park, North Carolina 27711
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, Duluth, Minnesota, 55804
1 To whom correspondence should be addressed. Fax: 919-541-4017. E-mail: gray.earl{at}epa.gov.
Received January 23, 2007; accepted March 12, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fungicide prochloraz (PCZ) induces malformations in androgen-dependent tissues in male rats when administered during sex differentiation. The sensitivity of fetal testicular steroidogenesis to PCZ was investigated to test the hypothesis that the reported morphological effects from maternal exposure were associated with reduced testosterone synthesis. Pregnant Sprague-Dawley rats were dosed by gavage with 0, 7.8, 15.6, 31.3, 62.5, and 125 mg PCZ/kg/day (n = 8) from gestational day (GD) 14 to 18. On GD 18, the effects of PCZ on fetal steroidogenesis were assessed by measuring hormone production from ex vivo fetal testes after a 3-h incubation. Lastly, PCZ levels in amniotic fluid and maternal serum were measured using liquid chromatography/mass spectroscopy and correlated to the inhibition of steroidogenesis. Fetal progesterone and 17
-hydroxyprogesterone production levels were increased significantly at every PCZ dose, whereas testosterone levels were significantly decreased only at the two high doses. These results suggest that PCZ inhibits the conversion of progesterone to testosterone through the inhibition of CYP17. To test this hypothesis, PCZ effects on CYP17 gene expression and in vitro CYP17 hydroxylase activity were evaluated. PCZ had no effect on testicular CYP17 mRNA levels as measured by quantitative real-time polymersase chain reaction. However, microsomal CYP17 hydroxylase activity was significantly inhibited by the fungicide (Ki = 865nM). Amniotic fluid PCZ concentrations ranged from 78 to 1512 ppb (207–4014nM) and testosterone production was reduced when PCZ reached 
500 ppb, which compares favorably with the determined CYP17 hydroxylase Ki (326 ppb). These results demonstrate that PCZ lowers testicular testosterone synthesis by inhibiting CYP17 activity which likely contributes to the induced malformations in androgen-dependent tissues of male offspring. Key Words: prochloraz; testosterone; CYP17; steroidogenesis; fetal testis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Development of the male reproductive system during gestation is dependent on androgen stimulation, and environmental chemicals that reduce androgen function have been shown to alter this development (Gray et al., 2006
; Sharpe, 2006). Some phthalates such as di(n-butyl) phthalate and di(ethylhexyl) phthalate (DEHP) disrupt male rat development by reducing fetal testosterone and insl3 production in the fetal testis (Mylchreest et al., 2002; Parks et al., 2000; Wilson et al., 2004). Although, it is unknown whether similar effects might be occurring in the human population, maternal levels of certain phthalates have been associated with reduced anogenital distance in male children, a marker of androgen function, which suggests these chemicals might affect human reproductive development (Swan et al., 2005).
The conazole pesticide prochloraz (PCZ), which is an agricultural fungicide, also disrupts male rat differentiation. Maternal exposure to PCZ during the gestational period of sexual differentiation resulted in hypospadias and other abnormalities, reduced reproductive organ weights, and increased retention of nipple/areolas in male rat offspring (Laier et al., 2006; Noriega et al., 2005; Vinggaard et al., 2005a). Since PCZ displays two anti-androgenic mechanisms, it is unclear which of these modes of action contributes to the altered development. First, PCZ is reported to reduce fetal testosterone production in vivo and ex vivo (Laier et al., 2006; Vinggaard et al., 2005a; Wilson et al., 2004). Secondly, PCZ is reported to be an androgen receptor (AR) antagonist in vitro and in vivo (Andersen et al., 2002; Noriega et al., 2005; Vinggaard et al., 2002).
Studies examining the effects of PCZ on fetal testosterone production used a limited dose range, precluding assessment of the PCZ-testosterone production dose-response relationship (Laier et al., 2006; Vinggaard et al., 2005a; Wilson et al., 2004). Additionally, the relationship between reduced fetal testosterone production and morphological effects in adult males has not been investigated. Furthermore, previous studies associating PCZ effects on male offspring measured fetal testosterone on gestational day (GD) 21, which would be after the testosterone surge within the fetal male (Huhtaniemi, 1994; Laier et al., 2006; Vinggaard et al., 2005b). These uncertainties make it difficult to assess whether the changes in androgen-dependent tissues in adult males due to maternal PCZ exposure are the result of decreased testosterone or AR antagonism. Therefore, this study first examined the sensitivity of fetal steroidogenesis during the testosterone surge to PCZ treatment by defining a five-point dose-response curve of ex vivo testosterone production. These data were compared to the reported dose-response of morphological effects in adult male rats (Noriega et al., 2005) to assess the correlation between them. Then we investigated the effects of PCZ on fetal CYP17 mRNA expression and CYP17 hydroxylase activity since this enzyme was viewed as a likely target for the anti-androgenic activity of PCZ. Steroidogenic acute regulatory (StAR) and CYP11A mRNA were also evaluated as potential targets of PCZ. Lastly, we measured amniotic fluid levels of PCZ to determine the concentration associated with reproductive malformations and to compare these values to the Ki values for CYP17 inhibition and AR antagonism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and dosing.
Timed pregnant Sprague-Dawley rats were shipped on GD 2–3 (GD 1 = day after mating) from Charles River Laboratories (Raleigh, NC) to the Environmental Protection Agency's Reproductive Toxicology Division animal facility. Animals were provided Purina Rat Chow 5008 and watered ad libitum. Environmental conditions were 22–23°C, 50–60% humidity, and a 14:10 light/dark cycle (lights on at 9:00 P.M.). Prior to dosing, animals were weight ranked and assigned to dose groups to minimize differences in means and variance among treatment groups. PCZ (CAS no. 67747-09-5, 99.5% purity by high-pressure liquid chromatography [HPLC]; Riedel-de Haën; Lot no. 2226x) was delivered to rats in corn oil (CAS no. 8001-30-7; Sigma Aldrich, St Louis, MO) in a volume of 2.5-ml/kg body weight. Dams were dosed daily from GD 14 to 18 with 0, 7.8, 15.6, 31.2, 62.5, or 125 mg/kg PCZ (n = 8 per dose). Calculated dose was determined by daily weight measurements. For microsome preparations, immature male Sprague-Dawley rats delivered from Charles River Laboratories on PND 23 were housed in the Environmental Protection Agency's Reproductive Toxicology Division animal facility. Animals were provided Purina Rat Chow 5001 and watered ad libitum. Environmental conditions were 21°C–24°C, 40–55% humidity, and a 12-h light/dark light cycle (lights on at 6:00 A.M.). The animal use protocol for this study was approved by the National Health and Environmental Effects Research Laboratory's Institutional Animal Care and Use Committee.
Ex vivo testis incubations.
On GD 18, dams were anesthetized with CO2, then decapitated between 0800 and 0945 h. Trunk blood was collected for serum progesterone and estradiol measurement. Fetal testis testosterone production was evaluated using a method described previously (Wilson et al., 2004). Briefly, the uterine tract was dissected and fetuses were quickly removed and placed in a tissue culture dish on ice. Each fetus was sexed under a dissecting microscope. The right and left testis were removed from the first three males of each litter and incubated individually at a humidified 37°C for 3 h in an Imperial III incubator (Barnstead/Lab-Line, Dubuque, IA). Each testis was placed in one well of a 24-well plate containing 500 µl of Gibco M199 media (no phenol red) in each well (Invitrogen, Carlsbad, CA) and gently rocked. The M199 media was supplemented with 10% dextran-charcoal-stripped fetal bovine serum (Hyclone, Logan, UT). After 3 h, media was removed and frozen at – 80°C in silicon-treated microcentrifuge tubes until hormone measurement. All hormones were measured using Diagnostic Products Corporation's Coat-A-Count kit (Los Angeles, CA).
Fetal testis gene expression.
The testes from the remaining males of each litter were removed for quantitative real-time polymerase chain reaction (qRT-PCR). The testes pooled by litter were immediately homogenized in Trizol reagent (Invitrogen) and frozen at – 80°C. Total RNA was extracted within 2–3 weeks using the method published by the manufacturer. Extracted RNA was digested for 30 min at 37°C with 1 IU Dnase I (Promega, Madison, WI) and stopped by heat inactivation (65°C for 5 min). RNA in the samples was quantified first using the Nanodrop spectrophotometer (Nanodrop technologies Inc., Wilmington, DE), and then by fluorescence with Ribogreen reagent (Invitrogen) using the manufacturer's method. DNased RNA (1 µg) was reverse transcribed using 4 µl of Promega 5x Improm buffer, 2.4 µl of MgCl2 (25mM), 1 µl dNTP (10mM), 0.5 µl RNAsin, 1 µl Promega Improm-II reverse transcriptase, and 1 µg random hexamer primers. Following reverse transcription, aliquots equivalent to 100 ng of cDNA were used for real-time PCR. Primer sequences for StAR, cholesterol side chain cleavage (CYP11A); steroid 17 alpha-hydroxylase, 17–20 lyase (CYP17); and a dual labeled fluorescent probe (Fam-Black Hole Quencher 1) (Table 1) synthesized by Intergrated DNA Technologies (Coralville, IA) were used. PCR reactions of 50 µl contained 2 µl dNTP (10mM), 10 pmol forward primer, 10 pmol reverse primer, 1.25 pmol Taqman probe, 1 µl Taq DNA polymerase (5 U/µl) (Invitrogen), 5 µl Promega Improm 10x buffer, diethyl pyrocarbonate (DEPC)-treated water, and varying amounts of 25mM MgCl2 (12 µl for StAR reaction, 12 µl for CYP11A, and 5 µl for CYP17). All samples were run in duplicate on a single plate using a Bio-Rad Icyler (Hercules, CA) and cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for 10 s were repeated 40 times. The threshold cycle was chosen to ensure all reactions were within the range where the amplification remained exponential. Copy number was determined from a standard curve generated by amplifying known cDNA quantities of each gene.
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CYP17 hydroxylase activity.
PCZ inhibition of CYP17 hydroxylase activity was assessed using testis microsomes prepared from adult Sprague-Dawley rats, under the assumption that CYP17 activity is similar between adult and fetal Leydig cells. On post-natal day (PND) 106, animals were anesthetized with CO2, then decapitated. The testes from each animal were removed and put into a cold 100mM potassium phosphate buffer solution (pH 7.4) containing 250mM sucrose and 1mM EDTA after removing the tunica. Testes were then homogenized using a Wheaton glass homogenizer and centrifuged for 10 min at 10,000 x g in a Beckman XL-90 centrifuge with Type 90Ti Rotor. The supernatant was removed and spun at 105,000 x g for 1 h. The resulting pellet was washed with fresh buffer and spun again at 105,000 x g for 1 h. The final pellet was resuspended in 100mM potassium phosphate buffer (pH 7.4) containing 20% glycerol and 0.1mM EDTA. Protein concentration was determined by Bradford assay using Bio-Rad Bradford Reagent (Hercules, CA). Enzymatic reactions with 0, 0.5, 1.0, 2.0, and 4.0µM PCZ were conducted in duplicate and the experiment was repeated (n = 2). Reaction solutions consisted of 100mM potassium phosphate buffer (pH 7.4), 50 µg protein, and NADPH-regenerating system (BD Gentest, Bedford, MA) for a total volume of 500 µl. The multiple concentrations of progesterone substrate included 1.1µM of [4-14C]progesterone (53 mCi/mmol activity, American Radiolabeled Chemicals, St Louis, MO). Reactions were stopped with 2 ml ethyl acetate at 10 min and placed in ice. Progesterone and its metabolites were extracted twice from the reaction mixture with 2 ml ethyl acetate, dried under nitrogen, and then spotted with a small volume of ethyl acetate onto a Whatman polyester-backed silica gel medium thin-layer chromatography (TLC) plate (Fisher Scientific, Pittsburgh, PA). Progesterone and metabolites were resolved using a 3:1 mixture of chloroform and ethyl acetate and identified by comigration of metabolite standards. Plates were then read using a Packard Instant Imager (Downers Grove, IL). Hydroxylase activity was calculated as the sum of the produced progesterone metabolites resolved on the TLC plates: 17
-hydroxyprogesterone, androstenedione, testosterone, and a minor metabolite believed to be 16-hydroxyprogesterone (Flück et al., 2003; Katagiri et al., 1998; Swart et al., 1993). The sum was used under the assumption that all progesterone metabolites were derived from CYP17 hydroxylase activity (e.g., androstenedione was produced only after hydroxylation of progesterone occurred). Michaelis constant (Km) and maximum velocity (Vmax) were determined from the double reciprocal plot (Lineweaver-Burke plot) of velocity–1 (1/Vo) at each substrate concentration–1 (1/[S]). The inhibitor binding constant (Ki) was then calculated from the negative x-intercept of the line produced from linear regression of Km/Vmax at each PCZ concentration using Graphpad Prism 4.0 software (San Diego, CA). The values for Km, Vmax, and Ki were the average of the two experimental runs.
Dosimetry.
Maternal serum and amniotic fluid (pooled by litter) were collected from half of the control and treated dams (0, 15.6, 31.3, 62.5, 125 mg/kg/day, n = 4 per dose) to determine PCZ concentrations. Samples were frozen at – 80°C until processed. Serum and amniotic fluid samples were weighed in the 2-ml centrifuge tubes in which they were stored, and then transferred (with acetonitrile rinse) to 15-ml polypropylene centrifuge tubes and diluted to 5 ml with acetonitrile. The empty 2-ml tubes were reweighed to determine sample weights. Samples were vortexed, sonicated for 30 min, vortexed again, and then placed in a refrigerator overnight. Samples were then centrifuged at 5°C and 1800 x g for 20 min, and the liquid portion was transferred into clean tubes and concentrated to approximately 0.5 ml in a warm water bath under a nitrogen stream. Each concentrate was diluted to 1.0 ml with 50/50 acetonitrile/water, vortexed, placed in ice for 2 h, and then centrifuged as before. Samples were transferred to 0.45-µm PTFE (polytetrafluoroethylene) spin filters and centrifuged at 10,000 rpm for 5 min. The filtrate was adjusted to 1.0 ml with acetonitrile/water and transferred to glass vials.
PCZ standards (obtained from Sigma, St Louis, MO) were prepared in 10% methanol/water, and spiked matrix samples were prepared by adding an aliquot of a PCZ solution (prepared in methanol) into control serum or control amniotic fluid (resulting in a nominal concentration of 953 ppb). The processed samples were placed into crimp top amber vials and analyzed for PCZ by reversed-phase HPLC with analyte confirmation by mass spectrometry (MS). The Agilent model 1100 HPLC (Wilmington, DE) consisted of a capillary pump, chilled auto sampler (4°C), heated column compartment (25°C), and a diode-array detector. An aliquot of sample (30 µl) was injected onto a Zorbax SB-C18 column (2.1 x 150 mm) (Agilent) and eluted isocratically with 80% methanol/water at a flow rate of 0.25 ml/min. PCZ concentrations were determined using the response at wavelength 220 nm or 230 nm and an external standard method of quantitation. An Agilent MS was used to confirm the presence of PCZ in the samples using an atmospheric pressure electrospray interface and selective ion monitoring acquisition of the molecular ion (positive polarity).
Statistical analysis.
Data were analyzed using the PROC GLM procedure from SAS (SAS v8, Cary, NC). Significant effects (p < 0.05) were further analyzed using LSMEANS (least squares means) to determine significance between the control and treatment groups. Hormone data from the right and left fetal testis of each male were averaged and then a litter mean was generated for analysis. Since testes were pooled by litter for RNA extraction, the individual data points were litter means. Heterogeneous data having SDs that increased with the means (e.g., hormone data) were log10 transformed for analysis to normalize variance. Correlation between amniotic and serum PCZ concentrations was analyzed by PROC CORR (Spearman) procedure from SAS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCZ treatment had no statistically significant effect on dam body weight at necropsy, but the pesticide did significantly decrease maternal weight gain (Table 2), which suggests some minimal maternal toxicity. Postimplantation loss (i.e., fetal loss) was not increased, indicating that PCZ did not increase fetal mortality at these doses. Maternal serum progesterone levels were unaffected by PCZ. Estradiol levels were significantly reduced at the 125-mg/kg/day treatment (Fig. 1). PCZ also significantly affected ex vivo fetal hormone production. Since progesterone and 17
-hydroxyprogesterone levels increased significantly at all doses, a no-observed-effect level could not be determined (Fig. 2A and 2B). PCZ decreased testosterone and androstenedione production but only at doses of 31.3 mg/kg/day and higher (Fig. 2C and 2D).
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The pattern of hormone production suggests that PCZ may inhibit conversion of progesterone to testosterone via CYP17. Reduced hormone production in the fetus could result from one of several mechanisms including inhibition of steroidogenic gene expression or from inhibition of enzyme activity. As suspected, PCZ did not alter testis gene expression. CYP17, StAR and CYP11A mRNA levels were unaffected by PCZ (Fig. 3). While PCZ did not alter the expression levels of these genes, it did inhibit in vitro CYP17 hydroxylase activity in a dose-dependent manner (Fig. 4A). The Km and Vmax values determined from the Lineweaver-Burke plot were 1.71µM (± 0.60 SEM) and 0.833 pmol/min/µg (± 0.149 SEM), respectively (Fig. 4B). The linear regressions generated for each concentration of PCZ intersected before the y-axis (1/[S] = 0.08 or [S] = 12.5µM). The increasing PCZ concentrations increased Km(app) and increased Vmax(app). The Ki from the Km/Vmax x-intercept was 865nM (± 240 SEM). Plotting the data in Hanes or Eadie-Hofstee plots did not aid in explaining the increased Vmax(app). Lower or higher substrate concentrations were excluded from analysis to determine if they contributed to the increased Vmax(app) but removing them did not affect the result. Additionally, when hydroxylase activity was calculated using only the 17
-hydroxyprogesterone metabolite, the increasing Vmax(app) result remained, which suggests that the other metabolites are not altering the slope. The intersection of the lines before the y-axis (Fig. 4B) may have to do with variation within the data and the increasing Vmax(app) is a product of this variation.
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The average recovery from six PCZ-spiked samples was 102.7% (± 35.1 SD), and the limit of detection was 20 ppb. PCZ concentration within amniotic fluid and maternal serum generally increased with dose and these values correlated well with each other (Spearman, Rs = 0.9611, p < 0.0001). The PCZ concentration within the amniotic fluid was generally about half that in the maternal serum, except at the highest dose implying that placenta might have been a barrier to PCZ distribution or there was metabolism by the fetus (Fig. 5A). PCZ was not detected in any of the control serum or amniotic fluid samples. The variability of PCZ concentration within treatment groups did not correlate with the order of necropsy, indicating that the variability was not due to time between dosing and necropsy. There was a negative correlation between ex vivo testosterone production and amniotic fluid levels (Spearman, Rs = – 0.4933, p < 0.0143). The lowest concentrations of PCZ in amniotic fluid that were associated with suppressed testosterone synthesis (
500 ppb) (Fig. 5B) compare favorably with the Ki of CYP17 hydroxylase activity for PCZ (326 ppb).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of the current study support the hypothesis that PCZ suppression of testosterone production contributes to the developmental effects of PCZ in the male fetus. Herein, this is the first study to show that PCZ inhibits testosterone production during the testosterone surge at the same dose levels as those that demasculinize the male rat in utero (Noriega et al., 2005
) (Table 3). The reported permanent morphological effects from gestational exposure to PCZ occurred at doses of 62.5 mg/kg/day and higher (Noriega et al., 2005) which is similar to the doses that significantly reduced ex vivo fetal testosterone production in this study. A low incidence of nipple retention in adult males was reported in the 31.3-mg/kg/day treatment group (Noriega et al., 2005) which corresponded to the nonstatistically significant 14% decrease in testosterone production, suggesting that nipple retention is highly sensitive to PCZ reduction in testosterone. More apparent than effects on androgen production were the increased levels of progesterone and 17-hydroxyprogesterone at all doses. There was no evidence that the elevated progestin levels contributed toward any fetal pathology, but this needs to be examined more thoroughly.
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In the present study, PCZ did not affect expression of CYP17, StAR, and CYP11A mRNA. These results are consistent with those of a previous PCZ study which found that these genes, among others, were not affected at GD 21 or PND 16 after maternal exposure (Laier et al., 2006
). These findings differ from those obtained with maternal di(n-butyl) phthalate exposure or DEHP exposure, compounds which produce anti-androgenic effects in male rats by inhibiting steroidogenic gene expression rather than directly inhibiting the enzymes (Barlow et al., 2003; Lehmann et al., 2004). The herbicide linuron is similar to PCZ in that it reduces testosterone production ex vivo (Hotchkiss et al., 2004; Wilson et al., 2004) and is a weak AR antagonist (Lambright et al., 2000; McIntyre et al., 2000), yet in utero linuron exposure induces epididymal and testicular lesions to a greater extent than PCZ and hypospadias/cleft phallus to a lesser extent than PCZ (Gray et al., 1999; McIntyre et al., 2000). The reason for the differences in adverse effects within male offspring, although both chemicals appear to work through similar mechanisms, is unknown.
The enzymatic activity of CYP17 was significantly affected in the present study. CYP17 hydroxylase converts progesterone to 17-hydroxyprogesterone, an intermediate which can disassociate from the enzyme or be further converted to androstenedione through CYP17 lyase activity (Tagashira et al., 1995). PCZ inhibited the hydroxylase activity (progesterone to 17-hydroxyprogesterone conversion), and the Ki is consistent with the Ki of several other imidazoles (Ayub and Levell, 1987). The lyase activity of CYP17 is also inhibited by PCZ, as suggested by the increase in 17-hydroxyprogesterone, and needs to be evaluated separately from the hydroxylase activity. How much of the PCZ inhibition of either the hydroxylase or lyase activity contributes to the reduced androgen production is not clear. Ketoconazole is suggested to primarily target the hydroxylase activity of CYP17 and only inhibit the lyase activity through competition with free 17-hydroxyprogesterone (Kuhn-Velten and Lessmann, 1992). Further work is needed to clarify how PCZ interferes with CYP17's hydroxylase activity and the lyase activity.
The amniotic fluid concentrations of PCZ at an effective dose for producing reproductive tract malformations were similar to the Ki for CYP17 hydroxylase inhibition. In contrast, the Ki value for AR antagonist activity of PCZ is considerably higher than the PCZ amniotic concentrations and Ki for CYP17 activity. The IC50 for PCZ is 60µM using cytosolic preparations from the rat prostate (Noriega et al., 2005). The estimated AR antagonism Ki of 20µM (Ki = IC50/1+([ligand]/Kd) [Cheng and Prusoff, 1973]; Kd = 0.5nM [Chang et al., 1988]), using the reported IC50, is considerably higher than the 4.0µM amniotic fluid concentration of PCZ in the 125-mg/kg/day treatment group. Together, these Ki values suggest that PCZ AR antagonism may be weaker than its CYP17 inhibition in the fetal male. However, one would need to also know what the testosterone and dihydrotestosterone levels were in the developing tissues to be more certain about the role of AR antagonism versus inhibition of testosterone synthesis inhibition by PCZ. It is possible that the reproductive effects in the male rat result are due to a cumulative effect between the two anti-androgen mechanisms. In utero exposure to 125 mg/kg/day PCZ results in a 12.5% incidence of cleft phallus/hypospadias (Noriega et al., 2005) which in the present study corresponded to a 25% drop in the ex vivo fetal testosterone production at 125 mg/kg/day. Similarly an 18% decrease in ex vivo fetal testosterone production by DEHP (not an AR antagonist) results in a low incidence of male reproductive tract malformations which increases to an incidence of 25% of males when ex vivo testosterone drops by 42% (Gray et al., unpublished data), suggesting that inhibition of testosterone synthesis by PCZ may play a major role in the development of reproductive tract malformations in male rats. However, more work is needed to model these effects and sort out the potential roles of these two mechanisms that disrupt the androgen-signaling pathway.
Maternal PCZ treatment causes a significant delay in parturition and at high-dose levels results in whole litter loss during delivery and some maternal death (Laier et al., 2006; Noriega et al., 2005; Vinggaard et al., 2005a). One of the maternal hormones critical in triggering parturition is estradiol. Here we show that maternal serum estradiol levels are reduced at GD 18, which may be indicative of PCZ affecting the estradiol increase leading up to parturition (Fang et al., 1996). Although it has been reported previously that PCZ inhibits aromatase in vitro (Andersen et al., 2002; Mason et al., 1987), our data here are the first to report a decrease of estradiol within rats.
In conclusion, the results of this study indicate that PCZ inhibition of testosterone production contributes to the reproductive malformations in the male rat after maternal exposure. The mechanism of reduced testosterone production does not involve down regulation of genes involved in steroidogenesis, but instead the direct inhibition of CYP17 enzyme activity.
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NOTES |
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Disclaimer: The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, ORD, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.
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ACKNOWLEDGMENTS |
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We would like to thank Dr Cynthia Rider and Mary Cardon for their help in fetal necropsies. We also would like to thank Carmen Wood for help with the qRT-PCR and Dr Christine McGahan for providing use of her Instant Imager. We would also like to thank Dr William Kelce for his comments on the manuscript. This research was supported in part by North Carolina State University and Environmental Protection Agency Co-op no. CT 826512010.
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