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BOR - Papers in Press, published online ahead of print January 26, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.031385
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BIOLOGY OF REPRODUCTION 72, 1324–1335 (2005)
DOI: 10.1095/biolreprod.104.031385
© 2005 by the Society for the Study of Reproduction, Inc.

Late Gestational Exposure to the Fungicide Prochloraz Delays the Onset of Parturition and Causes Reproductive Malformations in Male but Not Female Rat Offspring1

Nigel C. Noriega 2 , Joseph Ostby , Christy Lambright , Vickie S. Wilson , and L. Earl Gray, Jr. 

U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prochloraz (PZ) is an imidazole fungicide that displays multiple endocrine activities. It inhibits steroid synthesis via P450 modulation and acts as an androgen receptor (AR) antagonist, but its effects on male sexual differentiation have not been described. The purpose of the current study was to expand in vitro observations and to determine whether PZ affected sexual differentiation. PZ effects on AR-mediated gene expression were tested using a cell line (MDA-kb2) containing endogenous AR and stably transfected with an MMTV-luc reporter. PZ concentrations greater than 1 µM caused a dose-dependent inhibition of dihydrotestosterone-induced gene expression. PZ also inhibited R1881 binding to the rat AR (IC50 ~60 µM). In vivo, pregnant rats received PZ by gavage from Gestational Day 14 to 18 at doses of 31.25, 62.5, 125, and 250 mg/kg of body weight per day. PZ delayed delivery in a dose-dependent manner and resulted in pup mortalities at the two highest doses. In male offspring, anogenital distance and body weight were slightly reduced at 3 days of age. Additionally, female-like areolas were observed at 13 days of age at frequencies of 31%, 43%, 41%, and 71% in the lowest-dose to highest-dose groups, respectively. Weights of androgen-dependent tissues showed dose-dependent reductions. Hypospadias and vaginal pouches were noted in all males treated with 250 mg/kg, whereas those defects were observed in 12.5% and 6.25%, respectively, of males treated with 125 mg/kg. Treatment did not affect age of preputial separation in animals without penile malformations. Despite severe malformations in males, no malformations were noted in females. Together, these results indicate that PZ alters sexual differentiation in an antiandrogenic manner.

antiandrogen, environment, fungicide, hypospadias, male reproductive tract, parturition, penis, prochloraz, sexual differentiation, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prochloraz (1-{N-propyl-N-[2-(2,4,6-trichlorophenoxy) ethyl]} carbamoylimidazole), or PZ, is a broad-spectrum fungicide containing an imidazole moiety that has P450-dependent demethylase activity shared by many other imidazole and imidazole-like fungicides that inhibits the conversion of lanosterol to ergosterol in fungi. Imidazole fungicides can also affect activities of several P450-dependent enzymes in vertebrates, including aromatase [1, 2], 17{alpha}-hydroxylase, and 17,20 lyase activity [3, 4]. Although imidazoles are known to inhibit P450 enzyme activity in several systems, including rat fetal gonads, the developmental toxicity and multigenerational studies of PZ used in its risk assessment [5] did not detect malformations of androgen-dependent tissues in fetal or F1 males. Prior studies demonstrate that PZ is rapidly cleared. Approximately 99% of PZ administered orally to adult rats (at up to 250 mg/kg of body weight [BW]) is metabolized and excreted within 48 h with 2,4,6-trichlorophenoxyacetic acid, 2,4,6-trichlorophenoxyethanol, and 2,4,6-trichlorophenol as the major metabolites [6–8]. Neither PZ nor its metabolites 2,4,6-trichlorophenoxyacetic acid and 2,4,6-trichlorophenol affect androgen receptor (AR)-dependent gene expression in transfected cells [9].

In the fetal male rat, maternal PZ treatment inhibits testosterone production and increases progesterone production, indicating an inhibition of P450 17,20 lyase activity [10]. Although the P450 effects observed are to be expected from a member of the imidazole family of fungicides, PZ has also recently been shown to act as an AR antagonist in vitro and in vivo. This is a novel mechanism of action for this class of antifungals, which until recently, have not been shown to bind the AR [11]. In vitro, PZ inhibits AR-dependent gene expression in transiently transfected CHO cells [9]. In vivo, in a Hershberger assay (using castrated adolescent male rats with administered replacement testosterone), PZ exhibited antiandrogenic activity by reducing weights of androgen-dependent tissues (ventral prostate, seminal vesicles, levator ani plus bulbocavernosus muscles, and bulbourethral glands). Together, these data suggest that PZ has multiple mechanisms of endocrine action mediated through both the AR and several P450 enzyme pathways.

In the current study, we re-evaluated key observations from the literature by testing the ability of PZ to bind the rat AR and to modulate human AR-mediated gene transcription. We also evaluated effects of prenatal PZ exposure in the pregnant dam, and whether in utero PZ exposure affected the development of androgen-regulated tissues in male pups. We hypothesized that these tissues would be the most likely to display in vivo effects of PZ antagonism to androgen or aromatase activities [9, 12–15]. We expected that PZ would delay parturition if aromatase inhibition altered estrogen synthesis in vivo. In addition, we expected PZ to demasculinize male offspring (as do other AR antagonists) if antiandrogenic activity was expressed in the fetal male rat.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

We used prochloraz (45631; Riedel-deHaen; lot 9060X, CAS 067747-09-5, [C15H16Cl3N3O2] 99.4% pure by high-performance liquid chromatography, formula weight 376.67 g/mol), corn oil (C 8267; Sigma Chemical Co., St. Louis, MO; lot 81K2204, CAS 8001-30-7, density = 0.9 g/ ml), and dihydrotestosterone (DHT; Sigma; >99% purity). Hydroxyflutamide was provided by R.O. Neri (Schering Corp., Bloomfield, NJ).

AR-Dependent Gene Transcriptional Activation Assay

PZ, but not its metabolites, was reported to inhibit AR-mediated gene transcription [9]. We evaluated the effects of PZ on AR-mediated gene transcription using MDA-kb2 cells stably transfected with a reporter gene construct (pMMTV.neo.luc) [16]. Cells were maintained without CO2 at 37°C in L-15 media (Gibco BRL) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. AR-dependent transcriptional activation assays were conducted as described in [16]. Briefly, cells were plated at 5 x 104 cells per well in 96-well Costar luminometer plates and allowed to attach. Medium was replaced and cells were dosed with PZ (0.03–30 µM), DHT (0.1 nM), DHT (0.1 nM) + PZ (0.03–30 µM), DHT (0.1 nM) + hydroxyflutamide (1 µM) as an antiandrogen control, or vehicle control (ethanol) and incubated overnight. Cells were washed using Dulbecco PBS (14080-055; Gibco BRL) and lysed using 25 µl of E1531 lysis buffer (Promega, Madison, WI). Luciferase readings were measured using a Dyn-Ex MLX microtiter plate luminometer.

As a measure of cytotoxicity we determined the capacity of PZ to inhibit the ability of MDA cells to convert a tetrazolium salt (MTT) to blue formazan crystals as described in [17]. MDA cells were plated and PZ was administered with and without 0.1 nM DHT as described above for the transcriptional activation assay. Blue crystal formation was quantified using a Bio-Tek UV900 HDi microplate reader.

Binding Assay Using Homogenized Rat Prostate

The ability of PZ to compete with 1 nM [3H] R1881 for binding to the rat AR [9] was evaluated as described in [18] using ventral prostate tissue obtained from 90-day-old rats that had been castrated 24 h before tissue removal. Prostate tissue was homogenized on ice in low-salt TEGD buffer (1.5 mM disodium ethelynediaminetetraacetate, 1.0 mM phenylmethyl sulfonyl fluoride, 1.0 mM sodium molybdate, 1.0 mM dithiothreitol, 10 mM Tris, and 10% glycerol) at 10 ml per gram of tissue. Homogenates were centrifuged (30 000 x g) and supernatants were pooled.

PZ was used at concentrations of 1, 3, 10, 30, 100, and 300 µM; and 1 and 3 mM in duplicate during three separate runs. The assay mixture including PZ was incubated on a rotary mixer for 24 h at 4°C in 12 x 75 mm glass tubes with [3H] R1881 (83.5 Ci/mmol) and 10 µM triamcinolone acetate (to fully bind progesterone and glucocorticoid receptors). Incubations with a 100-fold molar excess of inert R1881 were used to estimate nonspecific binding. All incubations were performed using 300 µl of pooled prostate homogenate. Ligand-bound receptor was separated from unbound ligand using 500 µl of 60% hydroxyapatite slurry in 50 mM Tris buffer. Samples were washed three times in 50 mM Tris (with 600 times; g centrifugation between washes) to completely remove unbound ligand. Receptor-bound ligand was separated using 2 ml of ethanol and radioactivity (dpm) was determined using [3H] counting on a Beckman LS5000TD liquid scintillation counter.

Animals

Timed-pregnant Sprague-Dawley rats (approximately 90 days of age) were purchased from Charles River Laboratories (Raleigh, NC). Forty-eight rats (bred 9 September 2002) were used in the main study and 12 rats (bred 17 June 2002) were used in a preliminary dose range-finding study. Dams were delivered to Environmental Protection Agency facilities on Gestational Day (GD) 2 (the day after insemination was considered GD 1) and housed in transparent, 20 x 25 x 47 cm polycarbonate cages with laboratory-grade, heat-treated pine shavings (Northeastern Products, Warrensburg, NY). Animals were housed with a 14L:10D photoperiod (lights off at 1100 h) at 20–24°C and 40%–50% relative humidity. Dams were fed Purina Rat Chow 5008 ad libitum. Weaned pups were fed Purina Rat Chow 5001. Animals had 24-h access to filtered (5 µm) drinking water from the Durham, NC municipal supply. Water was tested monthly for Pseudomonas and every 4 mo for a suite of chemicals, including pesticides and heavy metals. The current study was conducted under a protocol that had been approved by the National Health and Environmental Effects Research Laboratory institutional animal care and use committee.

In Vivo Dose Range-Finding Pilot Study

A preliminary dose range-finding study using two dams per treatment group and three vehicle-treated dams was conducted to select dose levels for the large-scale study. Dams and pups were reared as described in the main study. Dams were gavaged from GD 14 to GD 18 using PZ doses of 0, 62.5, 125, 250, and 500 mg/kg BW per day with a dose volume of 2.5 ml corn oil/kg BW. The high dose was not used in the subsequent definitive study because delivery was delayed extensively. No pups with 500 mg/kg BW treatment in the pilot study survived to Postcoital Day (PCD) 35, and treatment inhibited maternal weight gain in the dams.

When offspring of treated dams were necropsied (males from PCD 114 to 139, females from PCD 297 to 338), all animals were examined for phallus abnormalities. Males were examined for nipple retention; and anogenital distance, glans penis, and gubernacular cord length were measured. Weights of pituitary, brain, ventral prostate, dorsolateral prostate, seminal vesicles (wet and dry), testes, epididymides, levator ani plus bulbocavernosus muscles, bulbourethral glands, adrenals, liver, and kidneys were also taken. In females, brain and pituitary weights were measured and reproductive tissues were examined for malformations. In the pilot study, the offspring data were analyzed using individual and litter mean values.

Main Study Treatment Assignment and Dosing

In an attempt to ensure that all dams were pregnant, the 8 (of 48) dams showing the smallest weight gain between GDs 3 and 13 were eliminated from the study. On GD 13, the rats were randomly assigned to treatment groups in a manner that provided each group with equal means and standard errors for body weight. Animals were assigned to treatment groups for a total of eight experimental blocks (each block contained one dam from each treatment group). Thus the final randomized block design consisted of five treatment groups with eight dams per group. Animals were distributed on cage racks according to experimental block (i.e., animals in the same block were housed in closest proximity).

Dams were dosed by gavage daily from GD 14 to GD 18 (fetal differentiation of androgen-dependent tissues) with PZ in corn oil at doses of 0, 31.25, 62.5, 125, or 250 mg/kg BW in a dose volume of 2.5 ml corn oil/kg BW. Treatment volume was calculated according to each dam's daily weight. Dams were dosed by gavage using a 1.5-inch x 20-gauge curved feeding needle (7910; Popper and Sons, Lincoln, RI) attached to a 1-ml glass syringe.

Parturition and Offspring

Normally, the Sprague Dawley rat has a gestation duration of about 22.5 days. In this study, we designated the scheduled delivery date of PCD 23 as Postnatal Day (PND) 1. On PCD 23 we repeatedly checked the litters to determine the time of complete parturition for each litter. Nursing dams with clean pups were considered to have completed delivery. Five sets of observations (at times 0720, 1130, 1340, 1650, and 2130 h) were made on PCD 23. Four sets of observations (at times 0700, 1115, 1200, and 1510 h) were made on PCD 24. Two sets of observations (at times 0700 and 0930 h) were made on PCD 25. The time of first observation was considered to be time zero. During subsequent observations, the numbers of delivered pups were noted. Animals showing vaginal bleeding or having pups scattered throughout the cage and uncleaned (blood on the pups and dam) or placentas in the bedding were considered to be in the process of delivery. In addition, signs of dystocia were also noted. To reduce observer effects on parturition the litter and nests were not disturbed, and the dams or pups were not handled.

Anogenital distance and body weight were measured in male and female pups on PCD 25 (PND 3). Measurements were conducted in a manner in which the observer was blind to the identity of the pups. Pups were manually restrained under a Leica MZ6 dissecting scope. Skin between the phallus and tail-base was extended maximally (using care not to stretch the skin) and anogenital distance measurements were made to the nearest 0.1 mm using a 10-mm ocular graticule. Scope magnification at 15x (1.5 x 10) was calibrated using a 1-mm stage micrometer with 0.01-mm divisions. After PCD 25, dead pups were removed from the mother's cage daily until the time of weaning. Partially cannibalized pups were counted as dead. The degree of necrosis in dead pups was highly variable. Dead pups were not examined further. On PCD 35, areola/nipple number and location were noted for each (male and female) pup in a blinded fashion. Pups were weaned (females on PCD 43 and males on PCD 44) and housed in unisexual groups of two to three littermates. Pups were placed in cages numbered and arranged in treatment blocks according to the assignment of the mother. Pups were given ear punch identification marks according to the mother's treatment, and picric acid marks to identify them within a litter. After pups were weaned, dams were killed using a CO2 anesthetic overdose, the uterus was removed, and uterine scars were counted to assess pup rearing success relative to the number of fetal implantations. Postimplantation loss was calculated as

{bire-72-06-01-eq1}

Beginning on PCD 46, female pups were examined daily for the vaginal opening. Pups were weighed on the day of vaginal opening. Beginning on PCD 61, male pups were examined for preputial separation (detachment of the prepuce from the glans penis). The prepuce was gently retracted far enough to note either the presence of a constriction at the base of the glans penis, or connective tissue that prevented the prepuce from being retracted to the base of the glans. Note that complete preputial separation (PPS) was distinguished from incomplete PPS in which portions of the prepuce remained attached to the glans, typically via a thread along the ventral midline of the phallus.

Necropsy of Mature Male Offspring

Female and male pups were necropsied separately. Beginning on PCD 147, males were necropsied in a randomized block design in which one male per litter per treatment block was selected at a time. One male was selected from each of the other treatment blocks before an additional male was selected from a given block. This process was continued until all males in the study were necropsied. Androgen-dependent organ weights were measured in each treatment block until three animals per litter were examined. This design ensured that differences in age at necropsy were accounted for across litters and treatments. Animals were anesthetized with CO2 before decapitation. All males were examined for vaginal pouches, the presence of nipples, and for phallus abnormalities including hypospadias, cleft phallus, or incomplete PPS. Androgen-dependent organs that were weighed included ventral prostate, dorsolateral prostate, seminal vesicles (with seminal fluid), testes, epididymides, levator ani plus bulbocavernosus muscles (combined), and glans penis. Kidney weights and liver weights were taken as diagnostics for general toxicological effects. The number of nipples at necropsy was compared against the number of areolas/nipples on PCD 35. Male necropsies were completed on PCD 213. Testes and epididymides from all males were preserved in Bouin fixative and placed in 70% ethanol after 24 h. Histopathologic examination of the testes and epididymides was conducted by Experimental Pathology Laboratories, Inc. (Research Triangle Park, NC), where tissues were sectioned at 4–6 µm and stained using hematoxylin-eosin.

Necropsy of Matured Female Offspring

Beginning on PCD 214, female offspring of control dams and those treated with 250 mg/kg BW per day were necropsied as described above until all high-dose animals had been examined. Because there was evidence of masculinized anogenital distance on female pups at PCD 25 (PND 3), indicators of masculinization, including anogenital distance, anovaginal distance, phallus length (defined here as the distance from the tip of the phallus to the anterior edge of the vaginal orifice), kidney to ovary distance, and vertical kidney to ovary distance [19] were measured. Wet and dry weights were recorded for the reproductive tract (oviducts, uterus, cervix, and vagina), ovaries, adrenals, liver, and kidney. As the examination of control and high-dose animals revealed kidney weight and body weight differences among treatment groups, all remaining control, low-dose, and mid-dose females were examined (using the remainder of the randomized blocks) for body weight, liver weight, and kidney weight. Female necropsies were completed on PCD 255.

Statistical Analysis

Data were analyzed using PROC GLM from SAS version 6.08 on the U.S. Environmental Protection Agency IBM mainframe computer. Statistically significant effects (F values) were further examined using the LSMEANS (two-tailed t-test) procedure to compare groups. If PZ demasculinized the male offspring, we expected to see a dose-related decrease in androgen-dependent tissue measures and increases in retained nipples and reproductive tract malformations. If the increase in anogenital distance at PCD 25 truly represented masculinization of the female offspring, we expected to see increases in reproductive tract malformations (vaginal agenesis, retained male sex accessory tissues) and reductions in the number of nipples. For in vivo data in the main study, although all measurements were recorded from individual animals (dams or pups), individual pup measurements were used to calculate litter means, and litter means were the unit of analysis. Thus the numbers per group are the number of litters (as opposed to number of pups). Significance for treatment effects on organ weights and anogenital distance data were determined using an analysis of covariance with body weight as the covariate.

For testicular histopathology analysis, vacuolization or atrophy of the seminiferous tubule epithelium was scored by Experimental Pathology Laboratories from 1 to 5, where a score of 1 was mildest and a score of 5 was most severe. We tested for treatment-related differences in percentages of affected animals using a chi-square statistic. Group-specific percentages of affected animals were compared using Fisher exact tests. In one analysis, animals receiving scores of 1 or higher were considered affected. In a second analysis, animals receiving scores of 2 or higher were considered affected.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro AR Transactivation and Binding

The ability of PZ to induce AR-mediated gene transcription was tested in MDA-kb2 cells containing endogenous human AR and stably transfected with an androgen-responsive luciferase reporter gene. Both hydroxyflutamide (1 µM) and PZ at doses of 3 µM or greater inhibited AR transcriptional activation induced by 0.1 nM DHT (Fig. 1A), with the effect of 1 µM hydroxyflutamide being roughly equivalent to the effect of 10 µM PZ. The IC50 for PZ effects on transactivation was between 3 and 10 µM. PZ was cytotoxic at the highest dose (100 µM) and reduced MTT activity in MDA-kb2 cells (P < 0.001; Fig. 1B). In the competitive binding assay, increasing concentrations of PZ inhibited [3H]R1881 AR binding with an IC50 of approximately 60 µM using cytosolic AR from rat ventral prostate tissue (Fig. 2).



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  		FIG. 1. In vitro assessment of PZ effects on androgen-regulated gene transcription and cytotoxicity in MDA-kb2 cells. X-axis numbers show PZ concentrations. Media indicates cell activity with no additions. EtOH shows cell activity with ethanol vehicle control. In all other treatments, the DHT dose was 0.1 nM. FLU indicates 1 µM hydroxyflutamide + 0.1 nM DHT. Luciferase data are expressed as fold induction over media levels. Asterisks indicate statistical difference compared to the 0 PZ (0.1 nM DHT) dose. *P < 0.05. **P < 0.001. Results are from three sets of assays run with four replicates each. Error bars = SEM. A) AR transcriptional activation. Luciferase activity, as fold induction over media, in transfected MDA cells treated with combinations of PZ and DHT. B) Cytotoxicity assessment of PZ + DHT combinations on MDA cells. Plate readings measure the conversion of MTT to formazan. Higher plate readings indicate higher numbers of live cells



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  		FIG. 2. PZ competitive inhibition of [3H]R1881 binding to the rat androgen receptor in cytosolic preparations from ventral prostate tissue. Results are from three sets of assays run in duplicate. Analysis was conducted using Sigmaplot Ligand software (available on the web at http:// www.statsol.ie/sigmaplot/ligand.htm). Error bars = SEM

Pilot Study Findings

Pilot study findings are summarized in Table 1. PZ treatment did not cause maternal death, but maternal weight gain during pregnancy was reduced in the 500 mg/kg-BW per day group (P < 0.01). There were no live births in the 500 mg/kg-BW per day group. The onset of parturition was delayed in all PZ-treated groups. Time to complete parturition was delayed up to 30 h in one of the dams that was treated with 125 mg kg-BW per day. At the same dose, another litter was entirely stillborn. Delays in delivery were associated with stillbirths at 125 and 250 mg kg-BW per day. Anogenital distance was reduced in male rat offspring at 62.5 and 250 mg/kg-BW per day and increased in females at 125 mg/kg-BW per day (significant using individual but not litter mean values). Males displayed areolas/ nipples on PCD 35 (PND 13) at frequencies of 40%, 71%, and 100% in the 62.5, 125, and 250 mg/kg-BW per day treatment groups, respectively. When necropsied as adults, the male offspring displayed reduced weight of the levator ani plus bulbocavernosus muscles in a dose-related fashion. At the time of necropsy, nipple retention in male offspring was observed at frequencies of 10%, 14%, and 100% in the 62.5, 125, and 250 mg/kg-BW per day groups, respectively. One-third of males in the 250 mg/kg-BW per day treatment group showed incomplete PPS, and the same percentage (including one animal with normal PPS) displayed a group of phallus abnormalities that included cleft phallus and hypospadias/epispadias.


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  		TABLE 1. Summary of pilot study results.a

Main Study: Maternal Effects of PZ

Although a dose-dependent reduction in weight gain was detected in dams between GD 14 and GD 22, the effects of treatment on weight gain were not statistically significant. Time to parturition increased significantly with dose, with dams in the 250 mg/kg-BW per day group initiating delivery more than 24 h after dams in other treatment groups. Dams at 125 mg/kg-BW per day or greater completed parturition more than 28 h after controls, all of whom delivered by time zero (0730 on PCD 23) (Table 2). Mean times to complete parturition were 2.5, 6.7, 22.2, and 28.4 h for 31.25, 62.5, 125, and 250 mg/kg-BW per day groups, respectively. Delivery times for dams dosed with 125 and 250 mg/kg-BW differed from times for dams dosed at 62.5 mg/kg-BW per day or lower (P ≤ 0.0005) but were not statistically different from each other (P = 0.1847). Five of eight dams treated with 125 mg/kg-BW per day delivered at least one stillborn pup. The rate of stillbirths at this dose range was 12% ± 4% (mean ± SEM) of the number of fetal implants. In the 250 mg/kg-BW per day dose group, one dam died during prolonged delivery and two dams did not deliver any live pups. The group of five remaining dams treated with 250 mg/kg-BW per day delivered stillborn pups at a rate of 32% ± 7% of the number of fetal implants.


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  		TABLE 2. Maternal PZ dosing effects on overall pup survivorship expressed as percentages (mean ± SEM) of live pups where the number of uterine implantation scars (according to litter) was 100%

Offspring Effects During Neonatal and Infantile Life

On PCD 25, body weights for pups of both sexes from treated dams were lower than control pups at 62.5 mg/kg-BW per day (female pups P = 0.03), 125 mg/kg-BW per day (male, P = 0.0019; female, P = 0.0002), and 250 mg/ kg-BW per day (both sexes, P ≤ 0.0001; Fig. 3). Effects on pup weight were transient and body weights did not differ among groups at weaning except for reduced weight in females dosed at 250 mg/kg-BW per day (P = 0.041).



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  		FIG. 3. Relationship of pup body weight at 3 days of age (PCD 25) to maternal dose. Error bars = SEM. Letters above bars show statistical groupings (P < 0.05) within the graph. Upper case letters refer to males, lower case letters refer to females. Groups sharing the same letter (e.g., A and AB) were not statistically different. Groups that do not share a letter (e.g., d and e) differed statistically (P < 0.05)

On PCD 25 male offspring from dams treated with PZ 125 or 250 mg/kg-BW per day had shorter anogenital distances compared with male controls (P = 0.006 and 0.0004, respectively), whereas female offspring in these two groups had larger anogenital distances compared with offspring of controls (P = 0.0369 and 0.0002, respectively). When anogenital distance was analyzed with body weight as the covariate, no treatment effects on anogenital distance were significant in the males, but females in the 250 mg/kg-BW per day group had longer anogenital distances compared with those of other groups (Fig. 4).



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  		FIG. 4. Relationship of anogenital distances at 3 days of age (PCD 25) to maternal dose. The data displayed on the left are adjusted anogenital distance values obtained when body weight was analyzed as a covariate, whereas the data on the right are unadjusted anogenital distance values. Error bars = SEM. Letters above bars show statistical groupings (P < 0.05) within the graph. Upper case letters refer to males, lower case letters refer to females. Groups sharing the same letter (e.g., A and AB) were not statistically different. Groups that do not share a letter (e.g., d and e) differed statistically

Postimplantation loss on PCD 25 was greater in the 250 mg/kg-BW per day group (P < 0.0001). Neonatal pup loss (pup loss between PCD 25 and PCD 35) was also increased in the 250 mg/kg-BW per day group (P < 0.0081). The percentage of offspring (as a proportion of the number of fetal implants) that survived to weaning was reduced in a dose-related manner by PZ treatment (Table 2). Dams dosed with 125 or 250 mg/kg-BW per day weaned fewer pups than controls (P ≤ 0.0348 and P < 0.0001, respectively).

Offspring Effects During Puberty and Adult Life

In the female offspring, pubertal landmarks, as well as age and body weight at vaginal opening, were not affected by prenatal PZ exposure. In the male offspring, 1 of 47 controls showed a persistent preputial thread (incomplete PPS) until PCD 82 (PND 60), but the prepuce was separated at the time of necropsy. Two of 46 (4.35%) 62.5 mg/ kg-BW per day-treated animals (from the same litter) showed incomplete PPS until PCD 82. A large preputial thread on one of the animals persisted to necropsy (PCD 169). In the 125 mg/kg-treated group, all males in one litter (four pups) and one male in another litter showed incomplete PPS until PCD 82, which persisted until necropsy (PCDs 147–213; Fig. 5). In all members of the 250 mg/kg-treated group, phalli were so malformed that preputial separation could not be fully assessed until necropsy. In pups with normal phallic morphology, there was no effect of treatment on age at separation at PZ doses of 125 mg/kg-BW per day or lower.



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  		FIG. 5. Phallus abnormalities in adult male rats receiving a 5-day in utero exposure to PZ. Panels are arranged from left to right according to dosage group indicated by numbers to the left of the diagram. A and B) Variation within controls. C and D) Animals with a maternal dose of 125 mg/kg-BW per day. E–G) Animals with a maternal dose of 250 mg/kg-BW per day. gl, glans penis; pr, prepuce; ur, urethral opening; os, os penis. Hypospadias is evident in the two highest dosage groups and (C–E) show examples of incomplete preputial separation. Severe phallus clefting and exposure of the os penis is evident in the highest dosage group (F and G)

Necropsy of Mature Male Offspring

All surviving males born to dams treated with 250 mg/ kg-BW per day retained nipples as adults. Permanently retained nipples were observed in males from all PZ-treated dosage groups, but the effect was statistically significant only at the highest dose. The mean number of nipples retained as adults were fewer than the number of areolas/ nipples noted on PCD 35 except in the highest-dosed group (Fig. 6). In all cases, inguinal areolas/nipples observed at PCD 35 were more likely to be transient, while thoracic and abdominal nipples were most likely to be retained in adults.



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  		FIG. 6. Prochloraz-induced areola/nipple retention in the male rat. Areolas and nipples were more prominent in infant male pups at PCD 35 (PND 13) than were permanent nipples in adult male rat offspring at necropsy (PCDs 147–213). Error bars = SEM. *P < 0.05 compared with the 0 PZ dose

In the 250 mg/kg-BW per day group, 100% of males showed cleft phallus and hypospadias, 70% also had an exposed os penis (Fig. 5), and 40% had a vaginal pouch (Fig. 7). Body weight at necropsy was reduced compared with that of controls, thus all organ weights were accordingly adjusted for body weight using the linear regression between body weight and the organ in question. Organs showing reductions in weight (adjusted) at 250 mg/kg-BW per day compared with those of controls were as follows: testes (P = 0.0052), epididymides (P = 0.0002), levator ani plus bulbocavernosus muscle (P = 0.0006), seminal vesicles (P = 0.02), and ventral prostate (P < 0.0001). Testes were typically flaccid and filled with fluid, whereas epididymides were incompletely formed and the corpus so reduced that caput and cauda were sometimes joined only by connective tissue (Fig. 8).



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  		FIG. 7. Prochloraz-induced vaginal morphology in adult male rats receiving a 5-day in utero exposure. A) Panoramic view of a vaginal pouch (forceps inserted) in a treated male from the 250 mg PZ/kg group. B) A close-up of a control female phallus and vaginal opening. C and D) Close-ups of vaginal pouches and phallus deformities in males from the 250 mg PZ/kg group. E–G) The most severely affected animal in the 125 mg PZ/ kg group. In one malformed male, an ejaculatory plug (F) was found embedded (E) in the vaginal opening (G)



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  		FIG. 8. Adult testicular and epididymal morphology from controls and a high-dose (250 mg/kg-BW per day) PZ-treated animal. Note that the testes of the treated animal are flaccid and the testicular artery appears malformed. Control vs. high-dose testis weights were 3.5 ± 0.08 vs. 3.0 ± 0.15 g (P < 0.05) and respective epididymal weights were 1.3 ± 0.02 vs. 0.99 ± 0.1 g (P < 0.05)

In male rats born to dams treated with 125 mg/kg-BW per day, 15.6% retained nipples, 18.75% showed incomplete PPS, 12.5% showed cleft phallus and hypospadias, 3.1% (one animal) had an exposed os penis, and 6.2% (two animals) had vaginal pouches. Weights of ventral prostate (P = 0.048) and epididymis (P = 0.011) were reduced. Unadjusted testis weights were reduced compared with those of controls (P = 0.0325; Table 3) but not when body weight adjustment was used (P = 0.0907).


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  		TABLE 3. Comparison (mean ± SEM) of organ weights (g) and testicular histopathology in male offspring of dams dosed with PZ from GD 14-18.a

At 62.5 mg/kg-BW per day, one male (2.2%) retained nipples and another showed incomplete preputial separation. Body weight-adjusted weight reductions were observed with the epididymis (P = 0.0105) and ventral prostate (P = 0.0478). One male (1.9%) at 31.25 mg/kg-BW per day retained nipples but no other reproductive malformations were observed in this group.

Treated animals showed testicular histopathology (Fig. 9) that included atrophy or vacuolization of the seminiferous epithelium (chi-square = 76.6, P < 0.01). Significant differences in percentages of affected animals compared with controls (P < 0.01) were observed in the three highest treatment groups (Table 3). Analysis of animals receiving a histopathology score of 2 or higher (chi-square = 36.5, P < 0.01) showed a dose-dependent increase in severity of the effect and percentages of animals in the 125 and 250 mg/kg-BW per day groups were significantly affected (P < 0.01) compared with that of controls. Testes were not always equally affected within individuals. At 250 mg/kg-BW per day the most severe histopathologies were unilateral.



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  		FIG. 9. Seminiferous tubule histological sections. A) Normal testis. B) Testes from an adult rat receiving a 5-day 250 mg/kg-BW per day gestational PZ exposure. Seminiferous tubules show atrophy and vacuolization. Bar = 100 µm

Necropsy of Mature Female Offspring

In females necropsied as adults, no differences were found between treatment groups in organ weights or in measurements of anogenital distance, anovaginal distance, phallus length (from anterior edge of vaginal orifice to phallus tip), kidney to ovary distance, or vertical kidney to ovary distance (Table 4). There was no indication of female masculinization at necropsy.


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  		TABLE 4. Parameter (mean ± SEM) comparisons in female offspring of control dams and dams dosed from GD 14-18 with 250 mg/kg-bw/day of PZ


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study we demonstrated for the first time that in utero PZ exposure induced permanent reproductive malformations such as retained nipples, hypospadias, cleft phallus, and vaginal pouch in male rat offspring. While the impact of these results on the risk assessment of PZ is uncertain, the determination that this fungicide is a teratogen can result in application of additional risk factors if PZ is deemed to be of special concern for the child. In this regard, reproductive malformations were not noted in the reproductive toxicity and teratology studies cited in PZ risk assessment documents [5, 8]. In these regulatory studies, there were no indications of any structural anomalies associated with treatment with dosage levels overlapping with those used in the current study. In contrast to the teratology studies that concluded that 85–100 mg PZ /kg-BW per day was without teratogenic effect, we found retained nipples, incomplete PPS, cleft phallus and hypospadias, vaginal pouches, and reduced ventral prostate, epididymal weights at 125 mg/kg-BW per day. Permanent reproductive alterations were also observed at 62.5 mg/kg-BW per day.

In the current study, we also demonstrated that in vitro PZ competed with a high-affinity synthetic androgen to bind to rat AR and that PZ also inhibited DHT-induced human AR-dependent gene expression at concentrations that did not induce cytotoxicity. Such results support the hypothesis that in vitro, PZ exhibits antiandrogenic activity by competing with androgens for receptor sites and support prior reports of PZ inhibition of AR transactivation [9, 14]. These results and findings from our laboratory demonstrating that PZ also inhibits fetal androgen production [10] indicate that PZ could interfere with the androgen signaling pathway during sexual differentiation via two independent mechanisms (i.e., by acting as an AR antagonist and by inhibiting testosterone synthesis). The AR affinity of PZ may distinguish it from other imidazole-like compounds. Although the imidazole-like compounds PZ, bifonazole, clotrimazole, econazole, isoconazole, ketoconazole, miconazole, and tioconazole all inhibit 17,20 lyase activity in testicular or adrenal microsomes [4], only PZ is an AR antagonist [11]. We also observed that PZ delayed parturition at all dosage levels, an effect consistent with in vitro studies [1, 13–15], demonstrating that PZ inhibits CYP19 aromatase, the enzyme required for estrogen synthesis.

As indicated above, the effects of PZ on sexual differentiation of androgen-dependent tissues could result from dual modes of action. PZ act as an AR antagonist and it inhibits testosterone production in the fetal testis [10]. Ex vivo testosterone production is significantly reduced in testes at GD 18 from male fetuses exposed in utero from GD 14 to GD 18, whereas progesterone and hydroxyprogesterone production are increased by as much as 10-fold [10]. Together, the results presented by Wilson et al. [10] and the current study indicate that PZ effects on fetal testes differed from effects of antiandrogens linuron and diethylhexyl phthalate, which decreased testicular testosterone production and left levels of progesterone unaffected. PZ-induced alterations in the steroidal milieu are consistent with the observation that 17,20 lyase may be among the most sensitive of the P450-dependent steroidogenic enzymes inhibited by some imidazoles [3].

While the developmental effects of PZ on weights of androgen-sensitive tissues in male offspring could result either from AR antagonism or from reduced testosterone synthesis, the profile of male reproductive tract malformations induced by PZ appears similar to effects observed with other classes of antiandrogens that are AR antagonists, such as flutamide or vinclozolin [20] but differs from profiles of the phthalate esters that inhibit fetal testosterone synthesis but do not bind AR [10, 21, 22]. The syndrome of male reproductive tract malformations produced by phthalates such as DEHP, DBP, and BBP [23, 24] includes high incidences of testicular, epididymal, and gubernacular agenesis, malformations that did not occur with PZ treatment in the current study.

The malformation profile of PZ-exposed males more closely resembles the profile observed with males exposed in utero to low doses of flutamide or intermediate doses of vinclozolin or procymidone [25–27]. Some effects (e.g., cryptorchidism) caused by higher doses of these three compounds were not observed in the current study with PZ. Taken together, these results suggest that high doses of PZ produce a profile of effects that closely resembles the profile produced by lower doses of flutamide. Flutamide is more potent at producing malformations than PZ, and administration of in utero PZ doses high enough to match the effects of flutamide appears unlikely to be achievable given the dystocia and high pup mortality associated with the 250 and 500 mg/kg-BW per day PZ doses in the current study. The profile observed with PZ at 250 mg/kg-BW per day also is similar to that observed in male rat offspring exposed to vinclozolin at 50 mg/kg-BW per day or procymidone at 100 mg/kg-BW per day [25, 26], in which androgen-dependent organ weight reductions and associated levels of hypospadias and nipple retention are similar among the three chemicals. With PZ, vinclozolin, and procymidone exposures, male malformations were produced without the severe testicular lesions observed with phthalate exposure [24]. Like PZ, procymidone [28] and vinclozolin metabolites [29] are AR antagonists, suggesting that AR antagonism may be a mechanism for malformation production through PZ exposure. Although further data are required before we can definitively determine what mechanisms of action are most responsible for the demasculinization of male pups, PZ antagonism of androgen in the Hershberger assay is consistent with in vivo AR antagonism by PZ.

As a rule, chemicals that disrupt the androgen signaling pathway during sexual differentiation disrupt the androgen-dependent tissue development in the fetal male without producing malformations in the female reproductive tract. This is true for phthalates that inhibit fetal Leydig cell hormone production [24], or the AR antagonists such as vinclozolin, procymidone, or flutamide [20] Androgens are required for male-specific tissues to differentiate from an indifferent state to the male phenotype. Reducing the androgen levels in the fetus or blocking androgen action at the level of the AR in the tissue induces a female-like phenotype. Because the pathway for androgen-induced tissue development is not activated during differentiation of the female reproductive tract, inhibition of the androgen signaling pathway does not affect the female phenotype. For this reason, administration of PZ during sexual differentiation produces severe malformations of androgen-dependent tissues in male rat offspring but is without obvious effect on the female rat. In contrast, administration of androgens to the female fetus induces malformations of the bipotential undifferentiated tissue. Androgens activate AR-dependent male-like differentiation, but treated male rats differentiate normally.

In the current study, administration of PZ from GD 14 to GD 18 delayed parturition at all dosage levels. PZ-induced aromatase inhibition [1, 13–15] and reductions in ovarian estrogen near term is a possible mechanism by which parturition was delayed.

Dystocia and delays in parturition are toxicities common to many conazole fungicides and, for some fungicides, these are the critical effects used to set no-observed-adverse-effect levels (NOAELs) in the risk assessments. For example, ketoconazole (which also targets P450 enzyme activity), like PZ, increased pup mortality and delayed parturition in dams dosed from GD 14 to GD 18 [30], but male offspring did not display signs of demasculinization. Similarly, the pyrimidine fungicide fenarimol also displays aromatase-inhibitory activity [31] and delays pup delivery [32], but in contrast to PZ, fenarimol did not produce overt effects on F1 male development [33].

The degree to which one form of endocrine toxicity predominates over the other (i.e., AR antagonism versus inhibition of aromatase) to disrupt reproductive function in the fetal male versus the pregnant dam depends to some degree on the duration of exposure. When PZ is administered at 250 mg/kg-BW per day or less for a brief period antiandrogenic effects are expressed along with delays in delivery that are not severe enough to reduce maternal or pup viability. However, had we expanded the dosing period to include the perinatal period it is likely that the effects on delivery would be more severe and occurred at lower dosage levels, which would preclude the survival of malformed pups. Because antiandrogenic effects of short-term dosing can be observed without mortality, the antiandrogenicity of PZ is likely more relevant to an acute risk assessment, compared with PZ effects on aromatase, which may be better examined in a longer-term, multigenerational study.

In addition to the current study, there are other reports of decreased gestational weight gain, delays in pup delivery, and loss of litters in dams in which PZ was administered during gestation [5, 8]. PZ may both inhibit and induce multiple P450 enzymes [34] and extended delivery times produced through PZ exposure may have resulted from reduced secretion of estrogens controlling the normal progression of pregnancy. Dose-related delays in parturition may be related to aromatase inhibition. Dystocia and pup retention during delivery likely accounted for most of the pup mortality in the current study. The ability to target multiple steroidogenic enzymes may allow PZ to simultaneously affect androgen- and estrogen-related physiology, and possible antiestrogenic activity of PZ has also been hypothesized to be mediated through P450 enzymes catalyzing estradiol hydroxylation [14].

The dose-related differences in female neonatal anogenital distances observed in both experiments in the current study could result from several endocrine mechanisms and could be interpreted as an indication of androgen-induced masculinization. However, no other treatment-related differences were found in infant or adult females to support this hypothesis. In addition, the differences in female anogenital distance were not permanent. If masculinized by an androgen, females would have been expected to show reduced areolas and nipple numbers; inhibited development of the vaginal orifice, cleft phallus, or the presence of prostate or other male sex accessory tissue [19], none of which were observed. Although PZ also has been shown to display possible estrogenic, antiestrogenic, or aryl hydrocarbon receptor (AHR)-mediated effects in vitro [9], it is not certain whether PZ displays these endocrine activities in the whole animal. Furthermore, exposure to chemicals that disrupt female rat sexual differentiation by acting as estrogen or AHR agonists produce reproductive tract malformations that were not present in the PZ-treated female offspring.

In vitro, PZ acts like estrogen to reduce Esr1 (estrogen receptor alpha [ER{alpha}] mRNA) and Esr2 (ERß mRNA) expression, as well as cause a transient reduction in ESR1 (ER{alpha} protein) expression in MCF7-BUS cells [35]. In contrast, PZ also inhibited estrogen-induced responses in cell proliferation and ESR1 transactivation assays [14]. Whether PZ could act as an estrogen agonist or antagonist could not be evaluated in the current study for several reasons. The fetal female reproductive tract will differentiate normally in the absence of ovaries or adrenal glands. Furthermore, mice lacking ESR1 or ESR2 develop a properly differentiated reproductive tract [36]. Tests of estrogen responsiveness on adult or pubertal female reproductive tissues would be necessary to assess whether PZ has estrogen agonist or antagonist activity in vivo. While in utero exposure to potent estrogenic drugs (personal communication) or steroids [19] at high-dose levels does increase anogenital distance in female rats at birth, this effect is associated with malformations of the external genitalia (cleft phallus with hypospadias) in female offspring, an effect not observed in the PZ-exposed female offspring.

Prochloraz has been reported to activate the AHR (also known as the dioxin receptor) [9] in vitro. However, in utero, AHR agonists produce a suite of reproductive tract effects that are distinct from those caused by in utero PZ treatment. Developmental effects of AHR agonists (i.e., 2, 3, 7, 8 tetrachlorodibenzo dioxin [TCDD]) in the male and female offspring differ markedly from the profile of effects seen in the current study. Effects of gestational TCDD exposure included female cleft phallus and persistent vaginal thread [37], delayed vaginal opening, and reduced ovarian weight [38] in female rat offspring. In male rat offspring, TCDD [39] causes epididymal agenesis and reduces sperm counts at low dosage levels, but unlike PZ, TCDD does not induce hypospadias or retained nipples in male offspring. Hence, the reproductive malformations induced by PZ in the current study are not a component of the dioxin profile of effects. Although the only effect observed herein in PZ-treated female offspring was a transient increase in neonatal anogenital distance, it is important to note that our evaluation of the female reproductive system was limited to an examination of a few developmental landmarks and organ weights, as the female offspring were not the focus of the current study, and it is possible that the reproducible, high-dose effect of PZ on neonatal anogenital distance is associated with adverse developmental reproductive toxicity on fertility, estrous cyclicity, behavior, or histopathology (or a combination of these) of the female rat.

Together, transcriptional activation, enzyme induction, and tissue incubation data suggest that PZ displays three distinct mechanisms of endocrine disruption, including AR antagonism [9], inhibition of aromatase activity [13], and inhibition of androgen synthesis [10].

Potential effects of the current study on PZ regulation have yet to be determined regarding human health, or fish and wildlife populations. That PZ causes malformations in the reproductive tract of male offspring at dosage levels that are not maternally toxic could have an effect on the acute dietary risk assessment of this chemical if a regulatory agency determined that these effects were displayed at dosage levels that were low enough to raise their level of concern about potential effects in humans. In addition, the observation that other pesticides such as vinclozolin [40] and procymidone [26] share a common mechanism of toxicity with PZ indicates that cumulative exposures to PZ with other pesticides might be important in an estimation of the risk of PZ or other antiandrogens to human health.

In summary, PZ is an androgen antagonist in vitro and in vivo. In the current study, we confirmed the original findings [9] that PZ binds to mammalian androgen receptors and inhibits AR-dependent gene expression in vitro. We also demonstrated that gestational exposure delayed parturition in the dam (consistent with previous unpublished reports), and showed for the first time that PZ causes reproductive malformations in androgen-dependent tissues of male offspring of exposed rats. PZ is the first imidazole fungicide shown to induce reproductive tract malformations.


   ACKNOWLEDGMENTS
 
Special thanks to Jonathan Furr and Cythia Wolf for assistance during necropsies.


   FOOTNOTES
 
1 The research described in this document has been reviewed by the U.S. Environmental Protection Agency (EPA) National Health and Environmental Effects Research Laboratory and does not necessarily reflect U.S. EPA views or policy. Back

2 Correspondence: Nigel Noriega, Endocrinology Branch, MD-72, RTD, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. FAX: 919 541 4017; noriega.nigel{at}epa.gov Back

Received: 28 May 2004.

First decision: 27 June 2004.

Accepted: 21 January 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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