ToxSci Advance Access originally published online on January 18, 2007
Toxicological Sciences 2007 97(1):65-74; doi:10.1093/toxsci/kfm004
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Published by Oxford University Press 2007.
Prochloraz Inhibits Testosterone Production at Dosages below Those that Affect Androgen-Dependent Organ Weights or the Onset of Puberty in the Male Sprague Dawley Rat
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* Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695
Reproductive Toxicology Division, Endocrinology Branch, National Human and Environmental Effects Research Laboratories, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599
1 To whom correspondence should be addressed at Reproductive Toxicology Division, Endocrinology Branch, MD-72, National Human and Environmental Effects Research Laboratories, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: gray.earl{at}epa.gov.
Received January 3, 2007; accepted January 10, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prochloraz (PCZ) is an imidazole fungicide that inhibits gonadal steroidogenesis and antagonizes the androgen receptor (AR). We hypothesized that pubertal exposure to PCZ would reduce testosterone production and delay male rat reproductive development. Sprague Dawley rats were dosed by gavage with 0, 31.3, 62.5, or 125 mg/kg/day of PCZ from postnatal day (PND) 23 to 42 or 51. There was a significant delay in preputial separation (PPS) at 125 mg/kg/day PCZ and several of the androgen-dependent organ weights were decreased significantly, but the significant organ weight effects were not consistent between the 2 necropsies (PND 42 vs. 51). At both ages, serum testosterone levels and ex vivo testosterone release from the testis were significantly decreased whereas serum progesterone and 17
-hydroxyprogesterone levels were significantly increased at dose levels below those that affected PPS or reproductive organ weights. The hormone results suggested that PCZ was inhibiting CYP17 activity. In a second pubertal study (0, 3.9, 7.8, 15.6, 31.3, or 62.5 mg/kg/day PCZ), serum testosterone levels and ex vivo testosterone production were significantly reduced at 15.6 mg/kg/day PCZ. In order to examine the AR antagonism effects of PCZ, independent of its effects on testosterone synthesis, castrated immature male rats were dosed with androgen and 0, 15.6, 31.3, 62.5, or 125 mg/kg/day PCZ for 10–11 days (Hershberger assay). In this assay, androgen-sensitive organ weights were only significantly decreased at 125 mg/kg/day PCZ. These data from the pubertal assays demonstrate that PCZ decreases testosterone levels and delays rat pubertal development, as hypothesized. However, the fact that hormone levels were affected at dosage eightfold below that which delayed the onset of puberty suggests that rather large reductions in serum testosterone may be required to delay puberty and consistently reduce androgen-dependent tissue weights. Key Words: prochloraz; testosterone; antiandrogen; puberty; Hershberger; steroidgenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evidence from wildlife studies and laboratory animal studies has raised concern that environmental chemicals might alter reproductive development in the human population (Colborn et al., 1993
). Environmental chemicals that reduce androgen function have been shown to alter male development in laboratory rats (Gray et al., 2006; Sharpe, 2006). The effects of antiandrogenic chemicals are dependant on the timing of exposure, and the periods of gestational and pubertal development are particularly susceptible (Sharpe, 2006; Stoker et al., 2000). Some chemicals can inhibit androgen function through multiple mechanisms (e.g., linuron [Gray et al., 1999; Lambright et al., 2000; Wilson et al., 2004]), and understanding the relevant mechanism of action is important to predict the subsequent consequences of exposure to gestational and pubertal reproductive development in humans and wildlife.
Prochloraz (PCZ) is an imidazole fungicide used in crop protection by inhibiting CYP51 leading to a weakened fungal cell membrane (Maertens, 2004; White et al., 1998). PCZ also interferes with androgen signaling through at least two mechanisms. First, PCZ has been shown to antagonize the human androgen receptor (AR) in transcription activation assays (Andersen et al., 2002; Noriega et al., 2005; Vinggaard et al., 2002) and in vivo it reduced the weights of androgen-sensitive tissues in the rat Hershberger assay (Vinggaard et al., 2002, 2005b). In addition to being an AR antagonist, PCZ also inhibits steroidogenesis. Gestational exposure to PCZ reduced fetal rat testosterone levels and increased progesterone levels ex vivo (Laier et al., 2006; Vinggaard et al., 2005a; Wilson et al., 2004). Exposure during sexual differentiation resulted in reproductive alterations in male rat offspring such as phallus abnormalities, reduced reproductive organ weights, and increased retention of nipples/areolas (Laier et al., 2006; Noriega et al., 2005; Vinggaard et al., 2005a). AR antagonism and/or reduced androgen levels are likely the primary mechanism of these effects on male reproductive development.
The effects of PCZ exposure during the period of pubertal development are not known. During this period, androgen action is necessary for development of the male reproductive system. Pesticides that alter androgen function have been shown to disrupt or delay this development. AR antagonists, such as vinclozolin and p,p'-Dichlorodiphenyldichloroetylene, significantly delay rat preputial separation (PPS) and development of androgen-sensitive organs (Kelce et al., 1995; Monosson et al., 1999). The purpose of this study was to test the hypothesis that pubertal exposure to PCZ delays pubertal development and to gain insight into the mechanism by which PCZ elicits these effects. Effects of PCZ on pubertal development were evaluated using the male pubertal rat assay that has been proposed as an alternate assay for detecting chemicals that alter androgen or thyroid function (EDSTAC, 1998). This protocol calls for a peripubertal exposure during which PPS is monitored, and effects on androgen-sensitive organ weights are recorded post-puberty. We modified this protocol (Stoker et al., 2000) by including measurement of in vivo and ex vivo testosterone and adding a necropsy at midpuberty, which is a time of rapid increases in androgen steroids (Monosson et al., 1999) and close to the day PPS is completed in the rat. These additional end points were added to determine if PCZ reduced testis testosterone production and serum testosterone at postnatal day (PND) 42/43 (midpuberty) and PND 50/51 (post-puberty). Since PCZ might disrupt pubertal development by dual mechanisms of action, we compared the dose-response relationships between the effects of PCZ on steroid hormone levels in the pubertal male rat with the effects in the Hershberger Assay. The Hershberger assay detects in vivo AR antagonism in a castrated immature androgen-treated male rat model (Gray et al., 2004; Owens et al., 2006) and therefore may allow us to assess the relative contribution between the two potential mechanisms of action.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and dosing solution.
Immature and immature castrated Sprague Dawley rats were delivered from Charles River Laboratories (Raleigh, NC) and housed in the Environmental Protection Agency's Reproductive Toxicology Division animal facility on PND 23 and PND 44, respectively. Animals were provided with Purina Rat Chow 5001 and watered ad libitum. Environmental conditions were 21–24°C, 40–55% humidity, and a 12L:12D light cycle (lights on at 0600 h). Animals in the Hershberger experiment were maintained on a 14L:10D cycle (lights on at 2100 h). Prior to dosing, animals were weight ranked and assigned to dose groups to minimize differences in means and variance among treatment groups. 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.
PCZ (CAS#: 67747-09-5, 99.5% purity by high-performance liquid chromatography; Riedel-de Haën; Lot# 2226x) was dissolved in corn oil (CAS# 8001-30-7, Sigma Aldrich, St Louis, MO) and administered by gavage for a final volume of either 2.5 or 5.0 ml/kg body weight. In the pubertal experiments, rats were given a dose volume of 5.0 ml/kg body weight from PND 23–30 due to the small size of the animal and then a dose volume of 2.5 ml/kg for the remainder of the study. In the Hershberger experiment, a 2.5-ml/kg volume was used throughout dosing. Animals from all the experiments were necropsied within 1.5–6.0 h of their final dose.
Pubertal experiments.
Immature Sprague Dawley rats were dosed daily by gavage with 0, 31.3, 62.5, or 125 mg/kg PCZ (n = 8–10/dose) from PND 23–42/43 or PND 23–50/51. This experimental design of two dosing periods allowed for measurement of chemical effects on hormonal and organ weight end points at mid- and post-puberty. The progression of PPS was inspected daily from PND 37 to necropsy at PND 42/43 or PND 50/51. The prepuce was gently retracted far enough to note the presence of either a constriction at the base of the glans penis or connective tissue that prevented the full retraction of the prepuce. Complete PPS was distinguished from incomplete PPS in which portions of the prepuce remained attached to the glans, typically via a midline thread located on the ventral side of the phallus. Selection of animals for each necropsy (PND 42/43 or 50/51) was random, rather than being based on PPS status, in order to avoid bias.
Organ weights were recorded at necropsy and trunk blood was collected by decapitation for serum measurements of testosterone, progesterone, 17-hydroxyprogesterone, androstenedione, estradiol, and luteinizing hormone (LH). In addition to serum measurements, ex vivo hormone production by the right testis of each animal was assessed. The right testis was removed, decapsulated, weighed, and then sectioned into 50–100 mg pieces. From each rat, two of the 50–100 mg pieces were incubated in oxygentated (95% O2:5% CO2) Gibco M199 media (Invitrogen, Carlsbad, CA) with 100 m IU/ml human chorionic gonadotropin (hCG; Sigma Aldrich) and two other pieces were incubated in the same media, but without hCG. Each piece was incubated in a 2.0 ml siliconized microcentrifuge tube for 1.5 h in a 34°C water bath shaker. The media was then collected by centrifuging the tubes at 4°C for 5 min at 1240 x g using a Beckman GS-6KR centrifuge. Collected media was stored at – 70°C for later quantification of progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. Samples were rethawed once for hormone measurements.
A second pubertal experiment was conducted with a wider dose range of 0, 3.9, 7.8, 15.6, 31.2, and 62.5 mg/kg/day (n = 6/dose) in an attempt to determine a no-effect level for serum testosterone and ex vivo testosterone production. The same methods and end points were used as in the first pubertal experiment except that animals were dosed from PND 23 to 42/43 and not PND 23 to 50/51. Data of completed PPS were not collected in this experiment since animal necropsy was at PND 42/43.
Hershberger experiment.
Male rats, castrated on PND 42, were dosed daily by gavage with PCZ (0, 15.6, 31.2, 62.5, or 125 mg/kg, n = 6/dose) from PND 49 to 58/59. Each rat received 100 µg testosterone propionate (TP) (Sigma Aldrich) by sc injection in 0.1 ml corn oil immediately after the gavage dose. A glass syringe and 25G 5/8'' needle was used for TP injection. Animals were anesthetized with halothane on the day of necropsy, and blood was collected via cardiac puncture to avoid contamination of blood with subcutaneous TP deposition. Testosterone and LH levels were measured in the serum, and organ weights were recorded.
Hormones.
Testosterone, androstenedione, 17-hydroxyprogesterone, and progesterone were measured in serum and media using Diagnostic Products Corporation's Coat-A-Count kit (Los Angeles, CA). The third generation estradiol radioimmunoassay (RIA) by Diagnostic Systems Laboratories (Webster, TX) was used for measuring serum estradiol. Serum LH was measured by RIA as previously described (Goldman et al., 1986) using materials supplied by the National Hormone and Pituitary Agency: iodination preparation I-6; reference preparation RP-3; and antisera S-11. Iodination material was radiolabeled with 125I (Dupont/New England Nuclear) by a modification of the chloramine-T method (Greenwood et al., 1963). All samples within a study were run together to avoid interassay variation.
Statistical analysis.
All data were analyzed using the PROC GLM procedure from SAS (SAS v8, Cary, NC). Significant effects (p < 0.05) were further analyzed using LSMEANS to determine significance between the control and treatment groups. In the pubertal studies, all PPS timing and weight data (except liver weight) were analyzed using the initial body weight (PND 23 weight before dosing) as a covariate to adjust for the fact that larger weanling male rats attain puberty at an earlier age than smaller rats (Stoker et al., 2000). In the first pubertal study, organ weights and hormone data from the two time points were analyzed by two-way ANOVA to determine treatment effects within the pooled data and determine if there was an age by treatment interaction. Additionally, PND 42/43 organ weight and hormone data from the first and second pubertal study were analyzed by two-way ANOVA to examine treatment effect over the pooled studies in order to determine how robust any PCZ effects were and if there was a study by treatment interaction. Organ weight data in the Hershberger study were analyzed using ANOVA. Analysis of liver weights in both the pubertal and Hershberger assays included necropsy weight as a covariate. Heterogeneous data having standard deviations that increased with the means (e.g., hormone data) were log10 transformed for analysis to normalize variance. If the hormone level of an individual sample was below the limit of detection, the lowest value on the standard curve was used for the statistical analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pubertal Experiment I
The first pubertal experiment was conducted to determine if PCZ delayed pubertal development and reduced testosterone levels. PCZ significantly delayed the initiation and completion of PPS in the highest dose group, 125 mg/kg/day (Table 1) by 1.1 and 1.8 days, respectively. Necropsies performed at PND 42/43 and PND 50/51 were used to compare effects on steroidogenesis and organ weights between these two time points. The levator ani plus bulbocavernosus muscle (LABC) and epididymides were decreased in weight at the PND 42/43 time point with greater sensitivity (i.e., affected at 62.5 mg/kg) than at the PND 50/51 time point when the seminal vesicle and ventral prostate were affected only at 125 mg/kg (Table 2). PCZ reduced body weight up to 7% at PND 42/43 indicating some minimal systemic toxicity, but this effect was not seen at the PND 50/51 necropsy. Kidney weights were significantly decreased in the higher doses and liver weights were increased, but the adrenal weight was only marginally affected by PCZ (Table 3).
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Serum testosterone was decreased dramatically by PCZ while treatment increased serum progesterone and 17
-hydroxyprogesterone compared to the controls (Figs. 1A and 1B). Serum androstenedione levels were generally below or close to limit of detection (0.15 ng/ml) and so were not analyzed. Serum LH was decreased in the 125-mg/kg/day dose group at the PND 50/51 time point, but this effect was not evident at PND 42/43 (Fig. 1D). Serum estradiol was unaffected by treatment (data not shown). In the ex vivo testis incubations, testosterone and androstenedione were significantly decreased and progesterone and 17-hydroxyprogesterone were increased compared to controls with and without hCG stimulation (Fig. 2). The steroid profile was the same at the PND 42/43 and PND 50/51 time points. Two-way ANOVA analysis found no treatment and age interaction in either the weight or hormone data. With the combined data from both time points, the ventral prostate, seminal vesicle, LABC, and epididymides were all significantly affected in the 125 mg/kg/day dose group, but not in the middose group of 62.5 mg/kg/day (data not shown).
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) is from tissue stimulated with hCG and dashed line (
) is from unstimulated tissue. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Pubertal Experiment II
A wider dose range with lower levels of PCZ was tested in the second pubertal experiment to define the dose response and no observable effect level (NOEL) of testosterone inhibition. Since the longer exposure period did not provide any enhanced sensitivity to PCZ, only the PND 23–42/43 dosing period was used. There were no significant effects on the LABC and epididymides weights at 62.5 mg/kg/day unlike in the first pubertal experiment (Table 2). The 3.9-mg/kg treatment group in the second pubertal experiment gained significantly more weight than the control (p = 0.0311) resulting in this group being heavier than controls at necropsy (Table 3).
The body weight at necropsy, LABC, and epididymides weight data from the first and second pubertal experiments were combined for a two-way ANOVA to determine if the combined data showed significant treatment effect and if there was an interaction between study and the organ weight data. The 62.5-mg/kg/day treatment had no statistical effect (p = 0.0834) on the combined epididymides weight and a statistically significant (p = 0.0199) effect on combined LABC weight, but there was no treatment effect on body weight. There was a significant study effect on LABC and body weight, but the interaction between study and organ weights was not significant. This suggests that although the mean body weight and LABC differed between studies, the treatment effect did not differ between the two studies. The lack of statistical significance at 62.5 mg/kg/day in the second study might be due to lower animal numbers and suggests that the organ weight effects at this dose are not robust. Liver weights increased significantly in the 62.5-mg/kg/day dose group, similar to the first pubertal experiment (Table 3). Serum progesterone and 17-hydroxyprogesterone did not increase in a similar fashion as in the first pubertal experiment. PCZ significantly reduced serum testosterone at 31.3 and 62.5 mg/kg/day, as in the first pubertal experiment, as well as at 15.6 mg/kg/day, a dose not tested previously (Fig. 1C). As in the first pubertal study, regardless of stimulation, testosterone and androstenedione levels were decreased in the ex vivo testis incubations while progesterone and 17-hydroxyprogesterone increased (Figs. 3A–D). The initiation of PPS was not delayed (n = 5–6), control PND = 40.0 ± 1.1 (SEM), 3.9 mg/kg/day = 39.2 ± 0.7, 7.8 mg/kg/day, 39.8 ± 0.6, 15.6 mg/kg/day 40.8 ± 0.8, 31.3 mg/kg/day 41.5 ± 0.4, and 62.5 mg/kg/day 41.2 ± 0.4, which is consistent with the first pubertal study. A NOEL of 7.8 mg/kg/day was determined for testosterone in the serum and ex vivo testis incubations and indicates that these measures had similar sensitivity to PCZ disruption (Fig. 4).
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) levels and ex vivo testosterone levels with hCG stimulation (Hershberger Experiment
Castrated male rats were treated with PCZ and TP to define the degree to which PCZ acted as an AR antagonist in vivo. At the highest dose group, PCZ significantly or marginally reduced the weights of several androgen-sensitive organs (Table 4), indicating that PCZ acts as an AR antagonist in vivo at this dose, but not at lower dose levels. Serum testosterone levels were not significantly different among the treatments. Serum LH was significantly decreased in the 125-mg/kg/day dose group, which is similar to the PND 50/51 time point of the first pubertal experiment. There was little overt toxicity in this assay. Body weight was not affected by treatment, but liver weights were significantly increased in the 31.3 mg/kg/day and greater doses. Kidney weights decreased similar to the first pubertal experiment, but this decrease was not statistically significant and there were no effects on adrenal weights (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we found that PCZ administered to the weanling male Sprague Dawley rat reduced ex vivo testis testosterone production and serum testosterone levels at doses eightfold lower than those that delayed puberty or robustly reduced androgen-dependent organ weights (i.e., 15.6 mg/kg/day vs. 125 mg/kg/day). The magnitude of the testosterone decrease in the first pubertal study without effects on PPS or organ weights raises the question of how pubertal development could appear unaffected when testosterone levels were so low. In fact, one purpose of the second pubertal study was to determine if the alteration of hormones could be replicated. Taken together, these data suggest that a large decrease in testosterone is needed to reduce androgen-sensitive organ weights and delay PPS. Since many of the tissues are dependent upon dihydrotestosterone (DHT) (Blohm et al., 1986
; George et al., 1989) produced locally from testosterone, it is possible that there was enough serum testosterone for conversion to DHT in all but the highest dose of PCZ. Measuring the amount of DHT and testosterone in the various reproductive tissues would help resolve the question of why particular organs were affected. Although the organ weights and PPS measured in this study were not affected by the same dose that decreased testosterone levels, other end points might be more sensitive to low testosterone.
The overall pattern of changes in hormone levels in the pubertal male rat suggests that the inhibition of activity associated with CYP17 is specifically responsible for the effects of reduced testosterone. In some instances, 17-hydroxyprogesterone levels were not elevated or marginally elevated at the high dose of 125 mg/kg/day. At this high dose, PCZ inhibition of CYP17's hydroxylase activity may be stronger than the inhibition of lyase activity. It is unlikely that PCZ inhibits the LH stimulation pathway since the pattern of inhibition in the ex vivo testis incubations did not change when the tissue was stimulated with hCG. Presumably PCZ acts like other imidazoles, which inhibit the CYP17 enzyme (Ayub and Levell, 1987). Ketoconazole has been reported to marginally affect puberty (Marty et al., 2001), presumably through inhibition of steroidogenesis even though hormone measurements showed no effect (Marty et al., 2001) or were not conducted (Ashby and Lefevre, 2000). The evidence for CYP17 inhibition is supported by our ex vivo measurements and may have been missed if only serum measurements were relied upon. Additionally androstenedione levels were generally undetectable in the serum, but the PCZ-induced alteration was easily measured in the ex vivo testis incubations. Experiments are currently being conducted to determine if CYP17 activity is competitively inhibited by PCZ in vitro (Blystone et al., in preparation), and these data will be included in a second manuscript describing the effects of PCZ on fetal hormone levels and expression of steroidogenic genes during sexual differentiation.
The ability of PCZ to antagonize the AR was assessed using the Hershberger assay. Androgen-sensitive weights were only affected at the highest dose (125 mg/kg/day), and our results differ from those that showed effects as low as 50 mg/kg/day in another Hershberger assay with Wistar rats (Vinggaard et al., 2002). The reason for the discrepancy is unknown, but may be due to differences in the two rat strains. Since PCZ and other conazole fungicides are known to affect the liver to varying degrees, it is possible that PCZ might decrease testosterone levels by increasing testosterone metabolism through induction of metabolic enzymes. However, this does not appear to be the case since serum testosterone levels were unaffected in the Hershberger assay.
The question of which "antiandrogenic" mechanism is responsible for delaying pubertal development is difficult to answer with these data. In fact, both mechanisms of action may be operative in vivo. A comparison of lowest observable effect levels among the present experiments (Table 5) shows that there is an overlap in doses that activate the different modes of "antiandrogen" action (i.e., AR antagonism and inhibition of testosterone production). The Hershberger assay provided insights into the AR antagonism, and there were clear effects at the 125 mg/kg dose. In the intact pubertal male rats, there may be a cumulative effect between the two mechanisms with the weak AR antagonism and strong steroidogenic inhibition of PCZ working together to delay pubertal development.
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PCZ demasculinzed the male rat offspring when pregnant rats received PCZ by gavage from gestational day 14–18 at doses of 31.3, 62.5, 125, and 250 mg/kg/day (Noriega et al., 2005
). Males displayed female-like nipples at 13 days of age at frequencies of 31%, 43%, 41%, and 71% in the lowest dose to highest dose groups, respectively. In addition, when necropsied as adults, F1 males displayed reduced weights and malformations in androgen-dependent tissues (125 and 250 mg/kg/day). Together these results indicate that PCZ alters sexual differentiation and delays puberty in an antiandrogenic manner at equivalent dosage levels. This suggests that male development, as measured by PPS and organ weights in the current study, may be similarly sensitive to PCZ during gestation and puberty. The ability of PCZ to alter fetal male rat hormone production levels is currently being evaluated in our laboratory to determine if the profile of effects is similar to that seen in the pubertal male rat herein and to determine if testosterone production is more sensitive in one developmental period than the other (Blystone et al., in preparation).
In addition to its effects on androgen synthesis, PCZ is also a potent inhibitor of aromatase in vitro (Mason et al., 1987; Vinggaard et al., 2000), and the delayed delivery seen in PCZ-treated dams (Noriega et al., 2005) suggests that it also inhibits estradiol synthesis in vivo. However, we were not able to detect a decrease in serum estradiol levels of PCZ-treated male rats in the pubertal studies. The lack of effect on estradiol herein may be due to the fact that the male normally produces such low levels of estradiol (pg vs. ng of the substrate, testosterone) and the abundance of testosterone may out-compete PCZ for the aromatase enzyme.
One endocrine effect in the current study was contrary to our expectation. We expected that PCZ would increase serum LH, possibly dramatically since it was expected to reduce serum testosterone levels and act as an AR antagonist. Either reducing testosterone dramatically or acting as an AR antagonist alone would be expected to elicit an increase in LH by eliminating the negative feedback of testosterone on the hypothalamic–pituitary axis. However, LH levels were unaffected in most PCZ doses in either the pubertal or the Hershberger experiments with the exception of decreased LH in the 125-mg PCZ/kg/day dose group. Other reports found that doses of PCZ that decreased androgen-dependent organ weights only increased LH at the highest dose of 250 mg/kg/day (Vinggaard et al., 2002), while in a second Hershberger study, LH did increase at 100 mg/kg/day (Vinggaard et al., 2005b). The data from our studies and previous studies indicate that in the LH, response to PCZ is highly variable and further investigation is warranted if this is to be clarified.
In summary, PCZ delayed pubertal development and reduced reproductive organ weights, but these effects were only apparent at a dosage level eightfold higher than the dose of PCZ that dramatically reduced testosterone levels in vivo and ex vivo. These data provide the first example of how a pesticide that is a steroid synthesis inhibitor affects PPS, organ weights, and testosterone end points in the pubertal male rat, an assay that is being considered for inclusion in the U.S. Environmental Protection Agency's (USEPA) Tier 1 screening battery for endocrine disrupters. Clearly, the sensitivity of the assay is enhanced by including the measurement of serum testosterone and ex vivo testis testosterone production. In addition, we were able to detect the changes in a shorter time period (20 vs. 30 days) (Stoker et al., 2000), and our assay required fewer animals per dose group (6–8 vs. 15) than recommended (EDSTAC, 1998). Based on these data we suggest that these testosterone measurements be included to detect steroid synthesis inhibitors like PCZ.
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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, Office of Research and Development, and 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 Emily Kaydos and Dr Tammy Stoker for their assistance with the LH assay. This research is supported in part by North Carolina State University EPA Co-op# CT 826512010.
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