Perinatal Exposure to the Fungicide Prochloraz Feminizes the Male Rat Offspring
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《毒物学科学杂志》
Department of Toxicology and Risk Assessment, Danish Institute for Food and Veterinary Research, DK-2860 Sborg, Denmark
Department of Chemistry, Danish Institute for Food and Veterinary Research, DK-2860 Sborg, Denmark
ABSTRACT
Prochloraz is a commonly used fungicide that has shown multiple mechanisms of action in vitro. It antagonizes the androgen and the estrogen receptors, agonizes the Ah receptor, and inhibits aromatase activity. In vivo prochloraz acts antiandrogenically in the Hershberger assay by reducing weights of reproductive organs, affecting androgen-regulated gene expressions, and increasing luteinizing hormone (LH) levels. The purpose of this study was to investigate reproductive toxic effects after exposure during gestation and lactation to prochloraz alone and a mixture of five pesticides (deltamethrin, methiocarb, prochloraz, simazine, and tribenuron-methyl). Prochloraz (30 mg/kg/day) or the mixture (20 mg/kg/day) was dosed to pregnant Wistar dams from gestational day (GD) 7 until postnatal day (PND) 16. Some dams were taken for cesarean section at GD 21, and others were allowed to give birth. Results showed that prochloraz and the mixture significantly reduced plasma and testicular testosterone levels in GD 21 male fetuses, whereas testicular progesterone was increased. Gestational length was increased by prochloraz. Chemical analysis of the rat breast milk showed that prochloraz was transferred to the milk. In males a significant increase of nipple retention was found, and the bulbourethral gland weight was decreased, whereas other reproductive organs were unaffected. In addition cytochrome P450 (CYP)1A activities in livers were induced by prochloraz, possibly as a result of Ah receptor activation. Behavioral studies showed that the activity level and sweet preference of adult males were significantly increased. Overall these results strongly indicate that prochloraz feminizes the male offspring after perinatal exposure, and that these effects are due, at least in part, to diminished fetal steroidogenesis.
Key Words: prochloraz; pesticide; antiandrogen; feminization; cytochrome P450 activity; behavior.
INTRODUCTION
An increase in adverse outcome of human and wildlife reproduction has been linked to chemical exposures. While the evidence for chemically induced adverse effects on wildlife reproduction is fairly good, it is still an open question whether there is a cause-and-effect relationship between human exposure to chemicals and reproductive health effects in males, including malformed sex organs, poor sperm quality, and increased incidence of testicular cancer. Recent epidemiological research has, however, indicated a causal relation between human exposure to pesticides and poor sperm quality (Swan et al., 2003) or increased incidence of cryptorchidism (Weidner et al., 1998).
Many chemicals have been demonstrated experimentally as being able to affect important endocrine processes, and some of these chemicals have been shown to act as antiandrogens (Gray et al., 1999a, 2001). The pesticides vinclozolin, procymidone, linuron, and the phthalates DEHP and DBP are among the best-documented antiandrogenic chemicals, because effects have been demonstrated both in vitro and in animal studies (Gray et al., 1999a, 1999b, 2001).
Prochloraz is an imidazole fungicide that is widely used in the industrialized world within horticulture and agriculture. The action of imidazoles used as fungicides or antimycotic drugs (e.g., ketoconazole) is based on the inhibition of the cytochrome P450–dependent 14-demethylase activity that is required for the conversion of lanosterol to ergosterol (Henry and Sisler, 1984), an essential component of fungal cell membranes. The molecular basis of this inhibition is the presence of an imidazole moiety that interacts strongly with the iron atom of cytochrome P450. The binding is fairly unspecific, and thus imidazole fungicides also inhibit the activities of a broad spectrum of other cytochrome P450-dependent enzymes, including key enzymes involved in biosynthesis and metabolism of steroids as for instance cytochrome (CYP) 19 aromatase (see references in Laignelet et al., 1992). Apart from inhibition, prochloraz is also capable of inducing some CYP enzymes (Laignelet et al., 1989; Needham et al., 1992).
In a recent in vitro study 22 pesticides, commonly used within agriculture and horticulture, were tested for effects on estrogen receptor (ER) and androgen receptor (AR) transcriptional activation, effects on proliferation of MCF7 breast cancer cells and for effects on CYP 19-aromatase activity (Andersen et al., 2002). Fourteen of the pesticides induced a significant response in at least one of the assays, and six pesticides showed positive response in more than one assay. One of these was prochloraz that turned out to have multiple mechanisms of action, as it antagonized both ER and AR and inhibited aromatase activity. In addition, prochloraz has been found to agonize the aryl hydrocarbon (Ah) receptor in vitro (Long et al., 2003). Recently, an in vivo Hershberger study showed that prochloraz acted as an antiandrogen at doses from 50 mg/kg, as weights of reproductive organs and expression of androgen-regulated genes in rat prostates were reduced, and serum LH levels were increased (Vinggaard et al., 2002).
The screening study by Andersen et al. (2002) also revealed that methiocarb acted as an ER agonist as well as an AR antagonist, and both deltamethrin and tribenuron-methyl stimulated proliferation of MCF7 cells. A follow-up in vitro and Hershberger study with a mixture of five pesticides having dissimilar modes of action including prochloraz, methiocarb, deltamethrin, tribenuron-methyl, and simazine, revealed additive antiandrogenic effects in vitro and slightly antiandrogenic effects in vivo (Birkhj et al., 2004). Simazine was included, as it has been found capable of inducing aromatase activity (Sanderson et al., 2000).
The purpose of the present study was, first, to investigate the reproductive toxic effects in male rat offspring after perinatal exposure to prochloraz. Second, we sought to test a mixture of the above-mentioned five pesticides, including prochloraz, to get an idea of the effects of a mixture of these pesticides that have both similar and dissimilar mechanisms of action. The selected end points differed in complexity, ranging from morphological and biochemical changes to changes in behavior of the animals (Fig. 1).
MATERIALS AND METHODS
Test compounds.
Prochloraz 99.4% pure (CAS no. 67747–09–5; N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-1H-imidazole-1-carboxamide), deltamethrin (CAS no. 52918–63–5), methiocarb (CAS no. 2032–65–7), and simazine 99.0% pure (CAS no. 122–34–9) were from LGC Promochem (Warsaw, Poland). Tribenuron-methyl 98% pure (CAS no. 101200–48–0) was from ChemService (West Chester, PA). The test compounds were dissolved in peanut oil (no. P-2144) from Sigma-Aldrich (St. Louis, MO). The saccharin used for the sweet preference test was >98% pure and from Sigma-Aldrich. NADPH was purchased from Aldrich Chemical Co. (Steinheim, Germany) and ethoxyresorufin, methoxyresorufin, pentoxyresorufin, benzyloxyresorufin, and resorufin were from Molecular Probes (Eugene, OR).
Animals and exposure.
Seventy-two time-mated, nulliparous, young adult Wistar rats (HanTac: WH, Taconic M&B, Ejby, Denmark) were supplied at day 3 of pregnancy. The animals were, upon arrival, randomly distributed in pairs and housed under standard conditions: semitransparent plastic-cages (15 x 27 x 43 cm) with Aspen bedding (Tapvei). They were housed in a room at a temperature of 22° ± 1°C and relative humidity of 55 ± 5%. The room was illuminated to give a cycle of 12 h light (9 p.m. to 9 A.M.) and 12 h of darkness (9 A.M. to 9 P.M.). A complete rodent diet for growing animals ALTROMIN 1314 (ALTROMIN GmbH, Lage, Germany) was given to pups until 6 weeks of age, after which ALTROMIN 1324 was given. Acidified tap water (to prevent microbial growth) was provided ad libitum. At the day after arrival, i.e., gestational day (GD) 4, animals were weighed and assigned to three groups of 24 animals with similar weight distributions. An acclimatization period of 4 days was allowed before starting exposure. The rats were gavaged with 0, 30 mg/kg prochloraz, or 20 mg/kg of the pesticide mixture from GD 7 to postnatal day (PND) 17. The pesticide mixture contained prochloraz, deltamethrin, methiocarb, simazine, and tribenuron-methyl that were administered orally in doses of 15.0, 1.25, 1.25, 1.25, and 1.25 mg/kg/day, respectively, i.e., a total dose of 20 mg/kg. Animals were divided into two sets, such that 36 animals representing all three groups were dosed 1 week prior to the next 36 animals. Initially it was planned to give the double dose of the mixture i.e., 40 mg/kg. However, after four doses were administered to half of the animals (replicate 1), clear signs of acute maternal toxicity were seen, and the mixture dose was therefore reduced to 20 mg/kg that was given from GD 11 to PND 16. The animals in replicate 2
Health status of dams and delivery.
The animals were housed in pairs until GD 20 and individually hereafter. The females were observed daily for signs of toxicity. Body weights were recorded on GD 4 and daily during the entire dosing period. The maternal weight gain from GD 7 to GD 21 and from GD 7 to PND 1 was calculated from the data. The first measure is based on the weights of the dams including the weight of the fetuses and may therefore, if affected, reflect an effect on the maternal animal and/or the fetuses. In contrast, the maternal weight gain from GD 7 to PND 1 (i.e., after birth) as well as the maternal body weight on PND 1 provides measures of the dam body weight only. The time of birth was monitored by checking the animals from GD 21 at 8 A.M., 12 P.M., and 4 P.M. Animals giving birth after 4 P.M. and before 8 A.M. were registered as having given birth at 12 A.M.
After delivery, weights of dams and individual pups were recorded. The pups were counted, sexed, and checked for anomalies. Pups found dead were investigated macroscopically for changes when possible. The day of delivery was designated PND 0 for the pups.
Cesarean sections GD 21.
Six dams from each group were randomly selected among the pregnant dams for cesarean section at GD 21. The dams were weighed and decapitated after CO2/O2 anesthesia, uteri were taken out, and the numbers of live fetuses, resorptions, and implantations were registered. Location in uterus, sex, nipples, and any anomalies of the offspring were recorded. Trunk blood was collected immediately after decapitation from all fetuses in heparin-coated vials for hormone analysis, and two pools per group were made from all male fetuses. Testes were taken for determination of hormone levels (stored in a –80°C freezer until analysis), testosterone production (processed immediately), and histopathology (fixed in Bouin's fixative [right testes] or in formalin [left testes]). The histopathological evaluation included a semi-quantitative investigation of Leydig cell hyperplasia, abnormal morphology of Leydig and Sertoli cells, number of germ cells, fusion of germ cell cytoplasm, and tubuli density, by scoring for normal (+) to mild changes (++) or moderate changes (+++).
Anogenital distance, nipple retention, and sexual maturation.
Anogenital distance in the offspring was measured at birth with a stereomicroscope. On PND 13 ± 1, all male and female pups were examined for the presence of aerolas/nipples, described as a dark focal area (with or without a nipple bud) located where nipples are normally present in female offspring. Sexual maturation was investigated by observing vaginal opening in the females and cleavage of the balanopreputial skinfold in the males. The latter was done by observing when the prepuce. which is initially fused to the glans penis, could be fully retracted (Korenbrot et al., 1977).
Section of pups PND 16.
From the control, prochloraz, and mixture group 16, 13, and 12 males and 16, 13, and 12 females, respectively, were randomly selected and killed at PND 16. Only 1 male and 1 female were taken from each litter, meaning that 16, 13, and 12 litters were represented on PND 16 from the three groups. All reproductive organs, livers, paired kidneys, and adrenals were weighed, and trunk blood was taken from the anaesthetized (CO2/O2) animals after decapitation. Serum was stored at –80°C for later hormone analyses. Livers were washed in phosphate buffered saline (PBS) and frozen in liquid N2 for CYP 450 analyses. Right and left testes were alternately fixed for histopathology in Bouin's fixative or in formalin (for 24 h) for immunohistochemistry.
Weaning.
The pups were weaned on PND 22 ± 1, and 16 pups per sex per group were kept for further testing in adulthood. As far as possible, only one male and one female from each litter were selected. The pups were randomly selected and were housed in pairs of the same sex and exposure status. The females who had not given birth and the dams were decapitated following CO2-anesthesia and examined macroscopically for changes and the number of uterine implantation sites.
Chemical analysis of breast milk.
Stomachs from female pups on PND 8/9 were excised 2 h or more after dosing of the mothers and frozen at –80°C until analysis. A total of 14, 13, and 9 samples (originating from 9, 8, and 6 litters) from the control, prochloraz, and mixture groups, respectively, were extracted and analyzed with an in-house validated method (modified from Poulsen and Granby [2000]). The samples of rat mother milk (between 0.1 and 0.5 g) were extracted with 20.0 ml of acetone for 2 min by an Ultra-Turrax mixer. Thereafter 20.0 ml ethyl acetate/cyclohexane 1:1 was added, and the extraction went on for additional 2 min. The extracts were centrifuged for 10 min at 3500 rpm. Aliquots of 25.0 ml were evaporated almost to dryness using 100 μl dodecane as a keeper, and diluted with ethyl acetate to 3.0 ml. Anhydrous sodium sulfate (1 g) was added, and the extracts were shaken in a Vibrofix for 1 min. A further 3.0 ml of cyclohexane was added and the extracts were shaken. The extract was filtered through Whatman No. 1 filter paper and cleaned up by gel permeation chromatography (GPC). The fraction containing prochloraz was added 100 μl dodecane and evaporated nearly to dryness and diluted with acetonitrile to 1.0 ml. Aliquots of 10 μl were analyzed with a LC-MS-MS (Quattro LC Triple Quadrupole from Micromass, Milford, MA). The mass spectrometer was operated with electrospray in the positive ion mode (ESI+). Detection was performed in Multiple Reaction Monitoring (MRM) mode. The MRM conditions for prochloraz were mass 375,66 > 307,66. The sample extracts from each analytical series were analyzed together and quantified using bracketing calibration curves at five concentration levels at 0.0087, 0.029, 0.087, 0.29, and 0.87 μg/ml prochloraz. The method was validated by double determination at five concentration levels, 0.13, 0.26, 0.43, 0.87, and 8.68 μg/g. Experiments were repeated three to five times. Recoveries were between 78% and 95%, and relative standard deviations of reproducibility were between 6.6% and 13.1%, depending on concentration level. The determination limit was 0.05 μg/g.
Hormone analysis.
Testosterone, progesterone, and luteinizing hormone (LH) were analyzed in serum from the pups at GD 21, PND 16, and PND 224. Testosterone and progesterone were extracted from the serum as previously described (Vinggaard et al., 2002; Birkhj et al., 2004) and the hormones were measured by time-resolved fluorescence using commercially available fluoroimmunoassay (FIA) kits (PerkinElmer Life Sciences, Turku, Finland).
Luteinizing hormone was analyzed at Turku University, Finland, using an immunofluorimetric assay as described by Haavisto et al. (1993).
Ex vivo testosterone and progesterone production at GD 21 was determined by decapsulating and incubating the testes in a shaking water bath at 34°C for 3 h in DMEM/F12 medium containing 0.1% BSA. Vials were centrifuged at 4000 x g for 10 min and supernatants were stored at –20°C until hormone levels were analyzed by the above-mentioned methods.
Cytochrome P450 activity analysis.
The ethoxyresorufin-O-deethylase activity (EROD) was measured in liver microsomes from PND 16 males and females using black microtiter plates (Black Optiplate, Packard). The reaction mixture contained 6 μg of liver microsomal protein and 0.1 M Tris–HCl (pH 7.8) containing 1.6 mg/ml BSA and 2 μM ethoxyresorufin (ER) in a total volume of 0.2 ml. The mixture was pre-incubated at 37°C for 2 min, and the reaction was initiated by the addition of 0.25 mM NADPH. Resorufin-associated fluorescence was measured using a fluorescence reader (Victor2 Wallac, PerkinElmer, Life Sciences, Denmark) employing an emission wavelength of 590 nm and a 560-nm excitation wavelength. The reaction was followed for 30 min with readings every 2 min, and the dealkylation rate was estimated on the basis of a resorufin standard curve ranging from 0 to 400 nM. Each sample was measured in duplicate and reaction rates were calculated. Benzyloxy- (BROD), methoxy- (MROD) and pentoxyresorufin-O-deethylase (PROD) activities were measured following the same procedures as described for the EROD activity.
It should be kept in mind that the dealkylation processes are only surrogate markers for CYP1A1, CYP1A2, CYP2B, and CYP3A, respectively. The authors are aware that some of the alkylresorufin substrates can be metabolized by several CYP isozymes, however, only the predominant CYP enzymes involved in the dealkylation reactions are listed above (Burke et al., 1994).
Section of adult males and semen quality analysis.
At PND 224 (32 weeks) adult males and females were killed and the same organs were taken out from the males as for PND 16 animals. The right testis was fixed in Bouin's fixative for histopathology, and the left testis was used for testosterone measurement.
Cauda of the right epididymis was used for motility analysis. Spermatozoa were collected by a modified Poke method (Slott and Perrea, 1993) and contained in Medium 199 Hanks & Hepes (Invitrogen Life Technologies, DK), supplemented with 0.5% bovine serum albumin (Sigma Chemical Company, St. Louis, MO). Sperm samples were analyzed by computer-assisted sperm analysis (CASA; HTM-IVOS version 12 Hamilton Thorne Research, Beverly, MA). The parameters evaluated were percent motile sperm and sperm velocities. Cauda of the left epididymis was frozen in liquid nitrogen for later sperm count. Cauda was thawed at room temperature, trimmed of fat, weighed, and homogenized in 1 ml Triton X-100. The homogenate was treated with ultrasonic sound and placed into a stain reaction vial containing DNA-specific fluorescent stain (Supra Vital IDENT Stain Kit, HTR). Sperm samples were placed in the HTM-IVOS. Samples were analyzed by CASA, counts were averaged, and the data were presented as number of sperm per gram cauda.
Behavioral studies.
The investigations were performed between 8.00 A.M. and 3 P.M. during the last of hour of the light cycle and during the dark cycle of the animals, i.e., their active period. The experimenter was kept unaware of the group to which an individual rat belonged. Exposed and control animals were tested alternately as were female and male animals. The ages for the assessment of end points were carefully planned and generally reflect that the same offspring were evaluated for all end points. For several end points, such as motor activity, Morris water maze learning, and sweet preference, the animals have to be sexually mature in order to find sexual dimorphic behavioral differences. In addition, the sweet preference test and the radial arm maze require single housing of the animals, and so these were the last tests to be performed.
Motor activity and habituation capability.
The activity of the animals was recorded in activity boxes with photocells for 10 x 3 min at PND 22 ± 1 prior to weaning, at PND 28 ± 1, and in adult animals at the age of 12 weeks. The total activity during the 30 min was used as a measure of general activity. In order to assess habituation capability, the 30 min was divided into two time periods of 15 min.
Play behavior.
The play behavior test was performed when the offspring were 34 ± 1 days old. The day before the test, the animals were housed individually in clean cages. Immediately before testing, the animals were placed in pairs with their usual littermate in their normal cage, and their behavior was recorded on videotape for 10 min. Afterwards, the latency to initiate play behavior, and the number of pinnings was scored. The test was performed in a room with no light, and behavior was recorded with a photosensitive camera.
Learning and memory (Morris water maze).
The animals were tested at the age of 13–14 and 17–18 weeks in a Morris water maze with a diameter of 220 cm as described earlier (Hass et al., 1995), with minor modifications. Four points on the rim of the pool, N, E, S, and W (not true magnetic directions), were used as starting points. A circular transparent platform was situated 1 cm below the water surface, and thus was invisible from water level. The animals were tested in four daily trials using the four starting points assigned in a pseudo-random sequence. The scheme used was: initial learning during 5 days; memory after 4 weeks for 2 days; reversal learning with the platform opposite the original location for 1 day; and new learning with the platform in the center for 1 day. A video-tracking device (Viewpoint video tracking system, Sandown Scientific, Middlesex, England) tracked the route of the animals; the latencies to find the platform, the path lengths, and swimming speeds were used as end points, as described in Hass et al. (1995).
Radial arm maze.
At the age of 22–27 weeks, the animals were investigated in a standard 8-arm radial arm maze as described elsewhere (Lam et al., 1991). One week before testing, the animals were housed one per cage and given restricted access to food in the afternoon (approximately 30 min per day). Once a day, they were given a few small pieces of the reward (peanut chips) to be used during testing in the radial arm maze. The food restriction continued throughout the testing period. The animals were tested daily in a total of 15 sessions over 3 consecutive weeks (5 trials per week). All eight arms in the maze were baited with peanut chips. In the daily test session, each rat was placed in the central maze area and allowed to explore the maze either until all chips were collected or until 10 minutes had elapsed. Latency to pass all the distally placed photoelectric cells and choice of arms was registered by computer. The number of errors, defined as visiting an arm that had already been visited, was calculated from the data.
Sweet preference test.
At the age of 21 weeks the animals were investigated in the sweet preference test. The week before testing, the animals were accustomed to drinking from two bottles with normal water. During testing, the animals were given the choice between normal water and water sweetened with 0.25% saccharin for 3 days. To avoid bias from preference of the left or right bottle, the placement of the bottles was equally distributed in the groups. The volumes of the solutions were determined each day. Body weight was registered in the same week and used for calculation of saccharin intake per 100 g body weight.
Statistical Analyses
Statistical evaluation of plasma hormone data, CYP 450 data, and semen data.
A one-way analysis of variance (ANOVA) was employed for all groups and, if significant, was followed by the post hoc test, Dunnett's test. Significance was judged at p < 0.05.
Sperm data were examined for normal distribution and homogeneity of variance. Single animal data were analyzed in a one-way ANOVA (proc glm) followed by Dunnett's t-test (version 8, SAS Institute Inc, Cary, NC).
Statistical evaluation of hormone levels and organ weights.
For statistical evaluation of testosterone and progesterone levels in testes (2–4 males per litter) and ex vivo testosterone and progesterone production at GD 21 (3–11 males per litter), all males were included in the analysis. Data from one male and one female per litter at PND 16 (12–16 litters per group) and one or two males per litter at PND 224 (11–16 litters per group leading to a total of 16 animals per group) were used to analyze terminal body weight and organ weights. To adjust for litter effects, litter was included in the ANOVA as an independent, random, and nested factor (proc mixed, SAS version 8, SAS Institute). Organ weights were analyzed using treatment as one main factor and age as another main factor, and body weight was used as a covariate. Non-processed and ln-transformed data were examined for normal distribution and homogeneity of variance. If an interaction between age groups and dose group was observed, the age groups were analyzed separately. When an overall significant treatment effect was observed, two-tailed comparison was performed using least squares means. In cases where normal distribution and homogeneity of variance were not obtained, data were additionally tested with the non-parametric Wilcoxon Scores, followed by a Kruskall-Wallis test. Effects on testis histopathology were analyzed by Fisher's exact test. A level of p < 0.05 was considered as statistically significant.
Statistical evaluation of pregnancy data, litter data, and behavioral data.
The litter was generally considered the statistical unit and the alpha level was 0.05. When more than one pup per litter is investigated, two closely related statistical strategies are available to avoid inflation of sample size (Holson and Pearce, 1992). The first alternative uses one score per litter, either a litter mean or the score of a single animal per litter. Second, and theoretically most attractive, one can include litter as an independent, random, and nested factor in the ANOVA. This approach specifically controls for litter effects and offers a direct statistical test of the significance of such effects. The two approaches are mathematically identical in their test for treatment effects (Holson and Pearce, 1992).
Data from the pre-weaning period were analyzed using litter means. Analyses of co-variance (ANCOVA) were used to test the differences between the groups in birth weights and pre-weaning pup weights while controlling for litter size. The remaining results were analyzed by ANOVA, when relevant, with nested design and repeated measures in trials or days. The SYSTAT PC-version software package was used for all computations (Systat 1990).
RESULTS
Pregnancy and Litter Data
Prochloraz had no effects on maternal weight gain during the period from GD 7 to GD 21 or on maternal weights on PND 1 (Table 1). However, a significant reduction in maternal weight gain from GD 7 to PND 1 was detected, which means that the prochloraz-dosed dams gained less weight than the control dams did. The mean pregnancy length was significantly increased by around 12 h after exposure to 30 mg/kg prochloraz. The control dams, as expected, all gave birth on GD 22 or GD 23 (6 and 10 dams, respectively). Among the dams exposed to prochloraz, none gave birth on GD 22; 15 on GD 23; and 1 on GD 24. Therefore, the mean increase in gestation length of 12 h reflects that birth is delayed by approximately 1 day for half of the animals. However, no effects on pup weight, sex ratio, litter size, postnatal death, or postimplantation-perinatal loss were detected. The pesticide mixture caused significantly reduced maternal weights after birth and decreased maternal weight gain from GD 7 to after birth. No effect on birth weight was found, but the body weights of the pups were significantly decreased at PND 6 and PND 13.
Chemical Analysis of Breast Milk
Chemical analysis of the content of prochloraz in rat's breast milk was performed by analyzing the milk content of stomachs from female pups sacrificed on PND 8/9. The results showed that no detectable levels were found in the control group (n = 6), whereas the concentration in the prochloraz and mixture group was measured to be 5.3 ± 3.9 μg/g (n = 13) and 1.5 ± 1.7 μg/g (n = 9), respectively.
On the basis of the pup weights at PND 0, 6, and 13 (Table 1), the weight gain of the pups was estimated to be 2 g/day during PNDs 6–9 and the body weight on PND 9 to be around 19 g. Assuming that the milk intake corresponds to approximately 2 ml/10 g body weight, the dose transferred at PND 9 to the pups via the breast milk can be estimated to be:
Thus, around 3% of the maternal dose of 30 mg/kg/day seems to be transferred to the pups on PND 9.
Anogenital Distance and Nipple Retention
Neither prochloraz alone nor the mixture affected anogenital distance in the male offspring at the selected dose levels (Table 1). A statistically significant effect on nipple retention at PND 13 was seen in the pups exposed to prochloraz, and the percentage of litters with nipples in male pups was significantly increased in both the prochloraz and the mixture group (Fig. 3). In the male fetuses at GD 21, no nipples or areolas were observed.
Hormone Levels GD 21, PND 16, and PND 224
Plasma testosterone, testicular testosterone and progesterone, and plasma LH were analyzed in GD 21 fetuses taken by cesarean section. The data showed that both prochloraz and the mixture significantly reduced plasma and testicular testosterone, whereas the testicular progesterone level was increased (Fig. 2). There was a slight tendency toward increased plasma LH levels, although this was not statistically significant. In PND 16 males serum testosterone, progesterone, and LH were not significantly affected, and in adult males (PND 224) no effects on testicular testosterone levels were found.
Basal testosterone and progesterone production ex vivo was investigated at GD 21 (Fig. 2). The reduced testosterone synthesis was statistically significant for the mixture group, but not for the prochloraz-dosed animals, whereas progesterone synthesis was significantly increased for both the prochloraz group and the mixture group.
Development of Pups (Organ Weights, Histology, and Sexual Maturation)
Body and organ weights for PND 16 males and females are shown in Table 2. The only significant effects that were detected were a decreased weight of bulbourethral glands caused by prochloraz, and a reduced body weight of males and females exposed to the mixture. The effect on body weight was also evident at PND 22, but only in the females at sexual maturation (Table 3). The day of sexual maturation as determined by vaginal opening and prepuce separation was unaffected in both male and female offspring. At PND 224 no effects on organ weights were seen for either prochloraz or the mixture (data not shown).
The testes were investigated macroscopically and microscopically by a blinded approach on GD 21, PND 16, and PND 224. At GD 21 moderate Leydig cell hyperplasia was observed in one of 10 males in the control group, in 6 of 18 males in the prochloraz group, and in 2 of 10 males in the mixture group. In a Fisher's exact test the increased incidence of Leydig cell hyperplasia was not statistically significant. At PND 16 and PND 224 no obvious treatment-induced histopathological effects were observed in the testis.
Cytochrome P450 Activities
The effect of prochloraz and the mixture on selected drug-metabolizing enzymes was investigated in livers of PND 16 males and females. From Figure 4 it is evident that prochloraz produced a minor induction of hepatic ethoxyresorufin O-dealkylase (EROD) and methoxyresorufin O-dealkylase (MROD) activities in both males and females, whereas neither penthoxyresorufin O-dealkylase (PROD) nor benzyloxyresorufin O-dealkylase (BROD) were affected. The mixture only significantly affected EROD and MROD in the females.
Semen Quality
Sperm counts were analyzed in PND 224 males. In the control, prochloraz, and mixture groups the sperm numbers were found to be 412 x 106 ± 68 (n = 16), 383 x 106 ± 60 (n = 16), and 375 x 106 ± 60 (n = 16), and no statistically significant effect was found. No effects on number of motile sperm cells or sperm velocities were detected either.
Behavioral Studies
Investigations of animal behavior comprised tests for play behavior, memory and learning, motor activity, and sweet preference. No exposure-related differences were found on play behavior or learning and memory functions, but the expected gender-related differences in behavior were found (data not shown). The motor activity was not affected in the prepubertal offspring, and no gender differences were seen. The latter finding was expected because gender differences in motor activity levels are usually not seen until after puberty (Blizard et al., 1975). The motor activity levels of the adult males and females were, as expected, statistically significantly different, with females exhibiting higher activity level than males. In the prochloraz-exposed adult males, a statistically significantly increased level of motor activity was found (Fig. 5). There was also a tendency toward a similar effect in the males exposed to the mixture, but the difference was not statistically significant (p = 0.064). The investigation of sweet preference showed, as expected, a clear sex difference, with females exhibiting higher preference for sweetened water than males. The males exposed to prochloraz generally showed a higher preference than control males during the 3 days of the test, but the differences were only statistically significant on the second day (Fig. 6).
DISCUSSION
Prochloraz was found to cause a small but statistically significant increase in mean pregnancy length of approximately 12 h. This result reflects that approximately half of the prochloraz animals gave birth 1 day later than expected. A similar effect was found by Noriega et al. (2003) after administration of higher doses of 62.5, 125, 250, or 500 mg/kg prochloraz to pregnant dams GD 14–18. In some high-dose–treated dams in that study, pup delivery was delayed up to 30 h. Previously, a classical 2-generation study with prochloraz was performed in rats (WHO, 1983). Prochloraz was given in the diet at 0, 37.5, 150, or 650 ppm (31 mg/kg) prior to mating and throughout mating, gestation, and lactation. An extended gestation period was also seen in this study, together with reduced food consumption, whereas no significant effects on mating performance or pregnancy rates were found.
The marked prochloraz-induced reduction in testosterone level and increase in progesterone level at GD 21 were both found in testis and plasma. In agreement with this finding, ex vivo progesterone production was significantly increased after prochloraz administration. However, no statistically significant effect on ex vivo testosterone production was found after administration of prochloraz 30 mg/kg alone. Wilson et al. (2004) have reported that, after administration of 250 mg/kg prochloraz between GD 14 and GD18, a reduced ex vivo testosterone production at GD 18 by around 50% was found, together with an increase in progesterone production (7.5-fold). In our study a dose of 30 mg/kg prochloraz was able to reduce fetal testosterone synthesis and increase fetal progesterone synthesis, indicating that the steroidogenic synthesis pathway in Leydig cells is directly affected by prochloraz. These changes in hormone levels are reversible, as we found no changes in testosterone and progesterone levels at PND 16 and no change in testosterone at PND 224.
It is conceivable that the delayed delivery of the dams, the increased progesterone levels, and the decreased testosterone levels in the fetuses are interconnected in some way. The increase of progesterone and the decrease of testosterone may be caused by an inhibition of 17-hydroxylase/17,20-lyase, 3-hydroxysteroiddehydrogenase and/or 17-hydroxysteroiddehydrogenase. As imidazole compounds have been shown to inhibit 17-hydroxylase and 17,20-lyase but not to have marked effects on the other two enzymes (Ayub and Levell, 1987), it is conceivable that this mechanism of action has played a major role. In the female rat the regression of the corpus luteum and a decline of progesterone secreted by corpus luteum in high amounts during pregnancy is a prerequisite for initiation and facilitation of delivery (Christenson and Deveto, 2003). Thus, although progesterone levels were not measured in the dams, the increased gestational length in prochloraz-dosed animals may be due to impaired luteolysis and increased progesterone levels.
A high plasma progesterone level in dams, leading to delayed parturition in rats, has been observed for fenarimol (WHO, 1995), a fungicide that resembles prochloraz in that it is both an aromatase inhibitor and an AR antagonist (Vinggaard et al., 2000, 2005).
Nipple retention was found in male pups exposed to prochloraz and, to a lesser extent, in pups exposed to the mixture. As the dose of prochloraz alone was twice as high as the dose given in the mixture, these findings indicate a dose–response relationship of prochloraz. No effects were found on the anogenital distance (AGD) of the male pups at birth. Both AGD and nipple retention are normally affected after prenatal exposure to antiandrogens (Gray et al., 2001). The lack of effect on AGD, together with a clear effect on nipple retention in the present study, indicates that nipple retention is a more sensitive end point than AGD after prochloraz exposure. The fact that increased nipple retention was also found by Noriega et al. (2003) after exposure GD 14–18 indicates that the underlying cause of this adverse effect involves factors taking place during gestation. Gray et al. (1999a) have described antiandrogens by categorizing several chemicals according to their pseudohermaphroditism index (PHI). One PHI score was calculated for effects on DHT-dependent tissues (AGD, areolas, retained nipples, ventral prostate size, hypospadias, and vaginal pouching) and one was given for effects on testosterone-dependent effects (seminal vesicle, epididymal, and testicular size or percent abnormal). The AR antagonists vinclozolin, procymidone, and p,p'-DDE all have the most pronounced effects on DHT-dependent tissues, whereas the phthalates that inhibit fetal steroidogenesis have the most marked effects on testosterone-dependent tissues. In the present study prochloraz exposure caused increased nipple retention and decreased bulbourethral gland weight, and we know that at higher doses the anogenital distance is reduced (Noriega et al. (2003) and own unpublished results). Because dihydrotestosterone (DHT) has been shown to be the proximal androgen for growth of the bulbourethral glands in mouse organ cultures (Cooke et al., 1987), these results indicate that DHT-dependent tissues are more severely affected by prochloraz. Although AR antagonism may be involved, the present data suggest that testosterone biosynthesis at the level of CYP 450 hydroxylase/lyase may mediate the observed DHT-mediated tissue alterations.
Prochloraz was found to induce the activity of CYP1A enzyme activities (EROD and MROD), but not BROD or PROD activities in PND 16 animals. This finding is in agreement with our previous result that prochloraz is an agonist of the Ah receptor (Long et al., 2003), leading to induction of CYP1A activities, whereas CYP2B activities were unaffected. Prochloraz has previously been reported to increase liver weights after administration of doses of 25 mg/kg and higher in castrated testosterone-treated rats (Birkhj et al., 2004; Vinggaard et al., 2002). This effect on liver weight may be a result of hypertrophy of liver cells, which may be caused by induction of CYP 450 activities. A previous study in which 250 mg/kg prochloraz was orally dosed to adult male rats for 3 days showed that both EROD (22-fold) and PROD (2.4 fold) were induced, which led to the suggestion that prochloraz is a mixed inducer of CYP 450 (Laignelet et al., 1989). In the present study we did not observe any effects on PROD, which we believe is due to the differences in dosages used in the two studies (30 mg/kg versus 250 mg/kg). It may be speculated that part of the antiandrogenic effect of prochloraz could be due to increased metabolism of testosterone via CYP 450 induction. However, as progesterone was increased, this seems not to be a plausible hypothesis.
In the present study no severe malformations of the reproductive system were observed. However, using this study design, in which animals were autopsied at various ages, may have decreased the power of the study compared to a design, in which all males are kept until adulthood and investigated for malformations, like the study reported by Foster and McIntyre (2002).
It is well known that there is a natural sex difference in the reproductive behavior of male and female rats. In addition, sex differences in a large number of other behavioral end points depend on early gonadal hormone secretion (MacLusky and Naftolin, 1981). Sex differences in central nervous function represent the outcome of interactions between several different factors, among which the hormones secreted by the gonads during development are of paramount importance. In mammals, the intrinsic behavioral pattern is generally female, with differentiation toward the masculine pattern of behavior occurring in the male as a result of exposure to testicular hormones during development. The conversion of testosterone into estradiol by aromatase in the brain, however, is critical for the organization of the male brain in rats (Hotchkiss et al., 2002). It has been shown in animal models that adult female rats have a higher spontaneous activity behavior than males and that they have a higher preference for sweetened water in a "sweet preference test" (MacLusky and Naftolin, 1981; Valenstein et al., 1967). Gender differences for spatial learning abilities have been reported in both humans and rodents, where males generally perform better than females (Roof, 1993). These gender-related differences were confirmed in the present study, including the activity and the sweet preference test. In these tests, we found that the prochloraz-exposed males in adulthood exhibited behavioral effects pointing toward incomplete masculinization/feminization of their behavior. It is conceivable that the feminized behavior of the male rats receiving prochloraz is a consequence of the markedly reduced testosterone levels found in this study during the late gestation period. Prochloraz has, however, also been shown to inhibit aromatase activity (Vinggaard et al., 2000), and this may have contributed to the effect by inhibiting the conversion of testosterone into estradiol, thereby reducing the estradiol levels needed for complete behavioral masculinization of the male rat brain.
In general, qualitatively similar, but apparently weaker effects were seen after exposure to the mixture compared to prochloraz administration alone. This was true for effects on hormone levels on GD 21, nipple retention, CYP 450 activities, and animal behavior. We believe that these effects of the mixture are due to the presence of 15 mg/kg prochloraz, which should be compared to the single dose of 30 mg/kg given in the prochloraz group. There are only few indications of differential effects between prochloraz and the mixture. These are the slight maternal toxicity of the mixture as seen from the reduced maternal weight on PND 1 and reduced weight of the pups after (but not before or at) birth, which suggests that pup growth is more vulnerable to the mixture during lactation than during gestation. Furthermore, a statistically significant reduction of the ex vivo testosterone production was seen for the mixture group, but not for the prochloraz group, and it may be speculated that the mixture component simazine, which has been reported to be an aromatase inducer (Sanderson et al., 2000), might have contributed to the lowered fetal testosterone levels. Overall, the results indicate that the relatively low doses of deltamethrin, methiocarb, simazine, and tribenuron-methyl present in the mixture did not markedly affect the response to prochloraz. The results further indicate that the non-AR active compounds (simazine and tribenuron-methyl) were not able to markedly modify the response caused by the AR-blocking compounds (deltamethrin, methiocarb, and prochloraz).
In conclusion, we have shown that prochloraz, apart from being able to block AR, is able to affect fetal steroidogenesis, two potential mechanisms of action for its antiandrogenic effects. These effects are suggested to be the cause of the increased nipple retention, the reduced weight of bulbourethral glands, and the feminized behavior of the male offspring. The adverse effects are subtle, but they occur after administration of a relatively low maternal dose of 30 mg/kg, and it was estimated that around 3% of this dose was transferred to the pups. It is of concern that a fungicide with these effects has been registered for use in many countries, and consideration should be given to reducing the risk of human exposure to this fungicide.
ACKNOWLEDGMENTS
This study was supported by the Danish Environmental Protection Agency and the Danish Research Council (grants no. 9801270 and 22–03–0198). We are indebted to Birgitte Mller Plesning, Rico Wellendorph Lehmann, Lisbeth Tranholm Andersen, Morten Andreasen, Lillian Sztuk, Bo Herbst, Ulla El-Baroudy, and Trine Gejsing for excellent technical assistance.
REFERENCES
Andersen, H. R., Vinggaard, A. M., Rasmussen, T. H., Gjermandsen, I. M., and Bonefeld- Jorgensen, E. C. (2002). Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicol. Appl. Pharmacol. 179, 1–12.
Ayub, M., and Levell, M. J. (1987). Inhibition of testicular 17-hydroxylase and 17,20-lyase but not 3-hydroxysteroid dehydrogenase-isomerase or 17-hydroxysteroid oxidoreductase by ketoconazole and other imidazole drugs. J. Steroid Biochem. 28, 521–531.
Birkhj, M., Nellemann, C., Jarfelt, K., Jacobsen, H., Andersen, H. R., and Vinggaard, A. M. (2004). The combined antiandrogenic effects of five commonly used pesticides. Toxicol. Appl. Pharmacol. 201, 10–20.
Blizard, D. A., Lippman, H. R., and Chen, J. J. (1975). Sex differences in open-field behavior in the rat: The inductive and activational role of gonadal hormones. Physiol. Behav. 14, 601–608.
Burke, M. D., Thompson, S., Weaver, R. J., Wolf, C. R., and Mayer, R. T. (1994). Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem. Pharmacol. 48, 923–936.
Christenson, L. K., and Devoto, L. (2003). Cholesterol transport and steroidogenesis by the corpus luteum. Reprod. Biol. Endocrinol. 1, 90.
Cooke, P. S., Young, P. F., and Cunha, G. R. (1987). A new model system for studying androgen-induced growth and morphogenesis in vitro: The bulbourethral gland. Endocrinology 121, 2161–2170.
Foster, P. M., and McIntyre, B. S. (2002). Endocrine active agents: Implications of adverse and non-adverse changes. Toxicol. Pathol. 30, 59–65.
Gray, L. E., Jr., Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R. L., and Ostby, J. (1999a). Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol. Ind. Health 15, 94–118.
Gray, L. E., Jr., Ostby, J., Monosson, E., and Kelce, W. R. (1999b). Environmental antiandrogens: Low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol. Ind. Health 15, 48–64.
Gray, L. E., Jr., Ostby, J., Furr, J., Wolf, C. J., Lambright, C., Parks, L., Veeramachaneni, D. N., Wilson, V., Price, M., Hotchkiss, A. et al. (2001). Effects of environmental antiandrogens on reproductive development in experimental animals. Hum. Reprod. Update 7, 248–264.
Haavisto, A. M., Pettersson, K., Bergendahl, M., Perheentupa, A., Roser, J. F., and Huhtaniemi, I. (1993). A supersensitive immunofluorometric assay for rat luteinizing hormone. Endocrinology 132, 1687–1691.
Hass, U., Lund, S. P., Simonsen, L., and Fries, A. S. (1995). Effects of prenatal exposure to xylene on postnatal development and behavior in rats. Neurotoxicol. Teratol. 17, 341–349.
Henry, M. J., and Sisler, H. D. (1984). Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi. Pesticide Biochem. Physiol. 22, 262–275.
Holson, R. R., and Pearce, B. (1992). Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species. Neurotoxicol. Teratol. 14, 221–228.
Hotchkiss, A. K., Ostby, J. S., Vandenbergh, J. G., and Gray, L. E., Jr. (2002). Androgens and environmental antiandrogens affect reproductive development and play behavior in the Sprague-Dawley rat. Environ. Health Perspect. 110, 435–439.
Korenbrot, C. C., Huhtaniemi, I. T., and Weiner, R. I. (1977). Preputial separation as an external sign of pubertal development in the male rat. Biol. Reprod. 17, 298–303.
Laignelet, L., Narbonne, J.-F., Lhuguenot, J.-C., and Riviere, J.-L. (1989). Induction and inhibition of rat liver cytochrome(s) P-450 by an imidazole fungicide (prochloraz). Toxicology 59, 271–284.
Laignelet, L., Rivière, J.-L., and Lhuguenot, J.-C. (1992). Metabolism of an imidazole fungicide (prochloraz) in the rat after oral administration. Food Chem. Toxicol. 30, 575–583.
Lam, H. R., Larsen, J.-J., Ladefoged, O., Mller A., Strange P., and Arlien-Sborg, P. (1991). Effects of 2,5-hexanedione alone and in combination with acetone on radial arm maze behavior, the "brain-swelling" reaction and synaptosomal functions. Neurotoxicol. Teratol. 13, 407–412.
Long, M., Laier, P., Vinggaard, A. M., Andersen, H. R., Lynggaard, J., and Bonefeld-Jrgensen, E. C. (2003). Effect of currently used pesticides in the AhR-CALUX assay: Comparison between the human TV101L and the rat H4IIE cell line. Toxicology 194, 77–93.
Maclusky, N. J., and Naftolin, F. (1981). Sexual differentiation of the central nervous system. Science 211, 1294–1303.
Needham, D, Creedy, C. L., and Dawson, J. R. (1992). The profile of rat liver enzyme induction produced by prochloraz and its major metabolites. Xenobiotica 22, 283–291.
Noriega, N. C., Gray, L. E., Jr., Ostby, J., Lambright, C., and Wilson, V. S. (2003). Prenatal exposure to the fungicide prochloraz alters the onset of parturition in the dam and sexual differentiation in male rat offspring. Toxicologist 72 (S1), Abstract 1375, p.283.
Poulsen, M.E., and Granby, K. (2000). Validation of a multiresidue method for analysis of pesticides in fruit, vegetables and cereals by GC/MS iontrap system. In Principles and Practices of Method Validation (A. Fajgelj and A. Ambrus, Eds.), Special Publication No 256 from The Royal Society of Chemistry, Cambridge, UK. ISBN 0–85404–783–2.
Roof, R. L. (1993). Neonatal exogenous testosterone modifies sex differences in radial arm and Morris water maze performance in prepubescent and adult rats. Behav. Brain Res. 53, 1–10.
Sanderson, J. T., Seinen, W., Giesy, J. P., and van den, Berg M. (2000). 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: A novel mechanism for estrogenicity Toxicol. Sci. 54, 121–127.
Slott, V. L., and Perrea, S. D. (1993). computer-assisted sperm analysis of rodent epididymal sperm motility using the Hamilton-Thorn Motility Analyzer. In Methods in Toxicology, Part A, Male Reproductive Toxicology (Chapin, R. E., and Heindel, J. J., Eds.), vol. 3, chapter 20, Academic Press, Inc., San Diego, CA.
Swan, S. H., Kruse, R. L., Liu, F., Barr, D. B., Drobnis, E. Z., Redmon, J. B., Wang, C., Brazil, C., and Overstreet, J. W. (2003). Semen quality in relation to biomarkers of pesticide exposure. Environ. Health Perspect. 111, 1478–1484.
Valenstein, E. S., Kakolewski, J., and Cox, V. C. (1967). Sex differences in taste preference for glucose and saccharin solutions. Science 1565, 942–943.
Vinggaard, A. M., Hnida, C., Breinholt, V., and Larsen, J. C. (2000). Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol. in Vitro 14, 227–234.
Vinggaard, A. M., Nellemann, C., Dalgaard, M., Jorgensen, E. B., and Andersen, H. R. (2002). Antiandrogenic effects in vitro and in vivo of the fungicide prochloraz. Toxicol. Sci. 69, 344–353.
Vinggaard, A. M., Jacobsen H., Metzdorff, S. B., Andersen, H. R., and Nellemann C. (2005). Antiandrogenic effects in short-term in vivo studies of the fungicide fenarimol. Toxicology 207, 21–34.
Weidner, I. S., Moller, H., Jensen, T. K., and Skakkebaek, N. E. (1998). Cryptorchidism and hypospadias in sons of gardeners and farmers. Environ. Health Perspect. 106, 793–796.
WHO, 1983, Pesticide Residues in Food—1983 Evaluations. Part II—Toxicology, p. 366–442. WHO/PCS/61, International Programme on Chemical Safety, World Health Organisation, Geneva.
WHO, 1995, Pesticide Residues in Food—1995 Evaluations. Part II—Toxicology, p. 75–104. WHO/PCS/96.48, International Programme on Chemical Safety, World Health Organisation, Geneva.
Wilson, V. S., Lambright, C., Furr, J., Ostby, J., Wood, C., Held, G., and Gray, L. E., Jr., (2004). Phthalate ester–induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis. Toxicol. Lett. 146, 207–215.(Anne Marie Vinggaard, Sof)
Department of Chemistry, Danish Institute for Food and Veterinary Research, DK-2860 Sborg, Denmark
ABSTRACT
Prochloraz is a commonly used fungicide that has shown multiple mechanisms of action in vitro. It antagonizes the androgen and the estrogen receptors, agonizes the Ah receptor, and inhibits aromatase activity. In vivo prochloraz acts antiandrogenically in the Hershberger assay by reducing weights of reproductive organs, affecting androgen-regulated gene expressions, and increasing luteinizing hormone (LH) levels. The purpose of this study was to investigate reproductive toxic effects after exposure during gestation and lactation to prochloraz alone and a mixture of five pesticides (deltamethrin, methiocarb, prochloraz, simazine, and tribenuron-methyl). Prochloraz (30 mg/kg/day) or the mixture (20 mg/kg/day) was dosed to pregnant Wistar dams from gestational day (GD) 7 until postnatal day (PND) 16. Some dams were taken for cesarean section at GD 21, and others were allowed to give birth. Results showed that prochloraz and the mixture significantly reduced plasma and testicular testosterone levels in GD 21 male fetuses, whereas testicular progesterone was increased. Gestational length was increased by prochloraz. Chemical analysis of the rat breast milk showed that prochloraz was transferred to the milk. In males a significant increase of nipple retention was found, and the bulbourethral gland weight was decreased, whereas other reproductive organs were unaffected. In addition cytochrome P450 (CYP)1A activities in livers were induced by prochloraz, possibly as a result of Ah receptor activation. Behavioral studies showed that the activity level and sweet preference of adult males were significantly increased. Overall these results strongly indicate that prochloraz feminizes the male offspring after perinatal exposure, and that these effects are due, at least in part, to diminished fetal steroidogenesis.
Key Words: prochloraz; pesticide; antiandrogen; feminization; cytochrome P450 activity; behavior.
INTRODUCTION
An increase in adverse outcome of human and wildlife reproduction has been linked to chemical exposures. While the evidence for chemically induced adverse effects on wildlife reproduction is fairly good, it is still an open question whether there is a cause-and-effect relationship between human exposure to chemicals and reproductive health effects in males, including malformed sex organs, poor sperm quality, and increased incidence of testicular cancer. Recent epidemiological research has, however, indicated a causal relation between human exposure to pesticides and poor sperm quality (Swan et al., 2003) or increased incidence of cryptorchidism (Weidner et al., 1998).
Many chemicals have been demonstrated experimentally as being able to affect important endocrine processes, and some of these chemicals have been shown to act as antiandrogens (Gray et al., 1999a, 2001). The pesticides vinclozolin, procymidone, linuron, and the phthalates DEHP and DBP are among the best-documented antiandrogenic chemicals, because effects have been demonstrated both in vitro and in animal studies (Gray et al., 1999a, 1999b, 2001).
Prochloraz is an imidazole fungicide that is widely used in the industrialized world within horticulture and agriculture. The action of imidazoles used as fungicides or antimycotic drugs (e.g., ketoconazole) is based on the inhibition of the cytochrome P450–dependent 14-demethylase activity that is required for the conversion of lanosterol to ergosterol (Henry and Sisler, 1984), an essential component of fungal cell membranes. The molecular basis of this inhibition is the presence of an imidazole moiety that interacts strongly with the iron atom of cytochrome P450. The binding is fairly unspecific, and thus imidazole fungicides also inhibit the activities of a broad spectrum of other cytochrome P450-dependent enzymes, including key enzymes involved in biosynthesis and metabolism of steroids as for instance cytochrome (CYP) 19 aromatase (see references in Laignelet et al., 1992). Apart from inhibition, prochloraz is also capable of inducing some CYP enzymes (Laignelet et al., 1989; Needham et al., 1992).
In a recent in vitro study 22 pesticides, commonly used within agriculture and horticulture, were tested for effects on estrogen receptor (ER) and androgen receptor (AR) transcriptional activation, effects on proliferation of MCF7 breast cancer cells and for effects on CYP 19-aromatase activity (Andersen et al., 2002). Fourteen of the pesticides induced a significant response in at least one of the assays, and six pesticides showed positive response in more than one assay. One of these was prochloraz that turned out to have multiple mechanisms of action, as it antagonized both ER and AR and inhibited aromatase activity. In addition, prochloraz has been found to agonize the aryl hydrocarbon (Ah) receptor in vitro (Long et al., 2003). Recently, an in vivo Hershberger study showed that prochloraz acted as an antiandrogen at doses from 50 mg/kg, as weights of reproductive organs and expression of androgen-regulated genes in rat prostates were reduced, and serum LH levels were increased (Vinggaard et al., 2002).
The screening study by Andersen et al. (2002) also revealed that methiocarb acted as an ER agonist as well as an AR antagonist, and both deltamethrin and tribenuron-methyl stimulated proliferation of MCF7 cells. A follow-up in vitro and Hershberger study with a mixture of five pesticides having dissimilar modes of action including prochloraz, methiocarb, deltamethrin, tribenuron-methyl, and simazine, revealed additive antiandrogenic effects in vitro and slightly antiandrogenic effects in vivo (Birkhj et al., 2004). Simazine was included, as it has been found capable of inducing aromatase activity (Sanderson et al., 2000).
The purpose of the present study was, first, to investigate the reproductive toxic effects in male rat offspring after perinatal exposure to prochloraz. Second, we sought to test a mixture of the above-mentioned five pesticides, including prochloraz, to get an idea of the effects of a mixture of these pesticides that have both similar and dissimilar mechanisms of action. The selected end points differed in complexity, ranging from morphological and biochemical changes to changes in behavior of the animals (Fig. 1).
MATERIALS AND METHODS
Test compounds.
Prochloraz 99.4% pure (CAS no. 67747–09–5; N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-1H-imidazole-1-carboxamide), deltamethrin (CAS no. 52918–63–5), methiocarb (CAS no. 2032–65–7), and simazine 99.0% pure (CAS no. 122–34–9) were from LGC Promochem (Warsaw, Poland). Tribenuron-methyl 98% pure (CAS no. 101200–48–0) was from ChemService (West Chester, PA). The test compounds were dissolved in peanut oil (no. P-2144) from Sigma-Aldrich (St. Louis, MO). The saccharin used for the sweet preference test was >98% pure and from Sigma-Aldrich. NADPH was purchased from Aldrich Chemical Co. (Steinheim, Germany) and ethoxyresorufin, methoxyresorufin, pentoxyresorufin, benzyloxyresorufin, and resorufin were from Molecular Probes (Eugene, OR).
Animals and exposure.
Seventy-two time-mated, nulliparous, young adult Wistar rats (HanTac: WH, Taconic M&B, Ejby, Denmark) were supplied at day 3 of pregnancy. The animals were, upon arrival, randomly distributed in pairs and housed under standard conditions: semitransparent plastic-cages (15 x 27 x 43 cm) with Aspen bedding (Tapvei). They were housed in a room at a temperature of 22° ± 1°C and relative humidity of 55 ± 5%. The room was illuminated to give a cycle of 12 h light (9 p.m. to 9 A.M.) and 12 h of darkness (9 A.M. to 9 P.M.). A complete rodent diet for growing animals ALTROMIN 1314 (ALTROMIN GmbH, Lage, Germany) was given to pups until 6 weeks of age, after which ALTROMIN 1324 was given. Acidified tap water (to prevent microbial growth) was provided ad libitum. At the day after arrival, i.e., gestational day (GD) 4, animals were weighed and assigned to three groups of 24 animals with similar weight distributions. An acclimatization period of 4 days was allowed before starting exposure. The rats were gavaged with 0, 30 mg/kg prochloraz, or 20 mg/kg of the pesticide mixture from GD 7 to postnatal day (PND) 17. The pesticide mixture contained prochloraz, deltamethrin, methiocarb, simazine, and tribenuron-methyl that were administered orally in doses of 15.0, 1.25, 1.25, 1.25, and 1.25 mg/kg/day, respectively, i.e., a total dose of 20 mg/kg. Animals were divided into two sets, such that 36 animals representing all three groups were dosed 1 week prior to the next 36 animals. Initially it was planned to give the double dose of the mixture i.e., 40 mg/kg. However, after four doses were administered to half of the animals (replicate 1), clear signs of acute maternal toxicity were seen, and the mixture dose was therefore reduced to 20 mg/kg that was given from GD 11 to PND 16. The animals in replicate 2
Health status of dams and delivery.
The animals were housed in pairs until GD 20 and individually hereafter. The females were observed daily for signs of toxicity. Body weights were recorded on GD 4 and daily during the entire dosing period. The maternal weight gain from GD 7 to GD 21 and from GD 7 to PND 1 was calculated from the data. The first measure is based on the weights of the dams including the weight of the fetuses and may therefore, if affected, reflect an effect on the maternal animal and/or the fetuses. In contrast, the maternal weight gain from GD 7 to PND 1 (i.e., after birth) as well as the maternal body weight on PND 1 provides measures of the dam body weight only. The time of birth was monitored by checking the animals from GD 21 at 8 A.M., 12 P.M., and 4 P.M. Animals giving birth after 4 P.M. and before 8 A.M. were registered as having given birth at 12 A.M.
After delivery, weights of dams and individual pups were recorded. The pups were counted, sexed, and checked for anomalies. Pups found dead were investigated macroscopically for changes when possible. The day of delivery was designated PND 0 for the pups.
Cesarean sections GD 21.
Six dams from each group were randomly selected among the pregnant dams for cesarean section at GD 21. The dams were weighed and decapitated after CO2/O2 anesthesia, uteri were taken out, and the numbers of live fetuses, resorptions, and implantations were registered. Location in uterus, sex, nipples, and any anomalies of the offspring were recorded. Trunk blood was collected immediately after decapitation from all fetuses in heparin-coated vials for hormone analysis, and two pools per group were made from all male fetuses. Testes were taken for determination of hormone levels (stored in a –80°C freezer until analysis), testosterone production (processed immediately), and histopathology (fixed in Bouin's fixative [right testes] or in formalin [left testes]). The histopathological evaluation included a semi-quantitative investigation of Leydig cell hyperplasia, abnormal morphology of Leydig and Sertoli cells, number of germ cells, fusion of germ cell cytoplasm, and tubuli density, by scoring for normal (+) to mild changes (++) or moderate changes (+++).
Anogenital distance, nipple retention, and sexual maturation.
Anogenital distance in the offspring was measured at birth with a stereomicroscope. On PND 13 ± 1, all male and female pups were examined for the presence of aerolas/nipples, described as a dark focal area (with or without a nipple bud) located where nipples are normally present in female offspring. Sexual maturation was investigated by observing vaginal opening in the females and cleavage of the balanopreputial skinfold in the males. The latter was done by observing when the prepuce. which is initially fused to the glans penis, could be fully retracted (Korenbrot et al., 1977).
Section of pups PND 16.
From the control, prochloraz, and mixture group 16, 13, and 12 males and 16, 13, and 12 females, respectively, were randomly selected and killed at PND 16. Only 1 male and 1 female were taken from each litter, meaning that 16, 13, and 12 litters were represented on PND 16 from the three groups. All reproductive organs, livers, paired kidneys, and adrenals were weighed, and trunk blood was taken from the anaesthetized (CO2/O2) animals after decapitation. Serum was stored at –80°C for later hormone analyses. Livers were washed in phosphate buffered saline (PBS) and frozen in liquid N2 for CYP 450 analyses. Right and left testes were alternately fixed for histopathology in Bouin's fixative or in formalin (for 24 h) for immunohistochemistry.
Weaning.
The pups were weaned on PND 22 ± 1, and 16 pups per sex per group were kept for further testing in adulthood. As far as possible, only one male and one female from each litter were selected. The pups were randomly selected and were housed in pairs of the same sex and exposure status. The females who had not given birth and the dams were decapitated following CO2-anesthesia and examined macroscopically for changes and the number of uterine implantation sites.
Chemical analysis of breast milk.
Stomachs from female pups on PND 8/9 were excised 2 h or more after dosing of the mothers and frozen at –80°C until analysis. A total of 14, 13, and 9 samples (originating from 9, 8, and 6 litters) from the control, prochloraz, and mixture groups, respectively, were extracted and analyzed with an in-house validated method (modified from Poulsen and Granby [2000]). The samples of rat mother milk (between 0.1 and 0.5 g) were extracted with 20.0 ml of acetone for 2 min by an Ultra-Turrax mixer. Thereafter 20.0 ml ethyl acetate/cyclohexane 1:1 was added, and the extraction went on for additional 2 min. The extracts were centrifuged for 10 min at 3500 rpm. Aliquots of 25.0 ml were evaporated almost to dryness using 100 μl dodecane as a keeper, and diluted with ethyl acetate to 3.0 ml. Anhydrous sodium sulfate (1 g) was added, and the extracts were shaken in a Vibrofix for 1 min. A further 3.0 ml of cyclohexane was added and the extracts were shaken. The extract was filtered through Whatman No. 1 filter paper and cleaned up by gel permeation chromatography (GPC). The fraction containing prochloraz was added 100 μl dodecane and evaporated nearly to dryness and diluted with acetonitrile to 1.0 ml. Aliquots of 10 μl were analyzed with a LC-MS-MS (Quattro LC Triple Quadrupole from Micromass, Milford, MA). The mass spectrometer was operated with electrospray in the positive ion mode (ESI+). Detection was performed in Multiple Reaction Monitoring (MRM) mode. The MRM conditions for prochloraz were mass 375,66 > 307,66. The sample extracts from each analytical series were analyzed together and quantified using bracketing calibration curves at five concentration levels at 0.0087, 0.029, 0.087, 0.29, and 0.87 μg/ml prochloraz. The method was validated by double determination at five concentration levels, 0.13, 0.26, 0.43, 0.87, and 8.68 μg/g. Experiments were repeated three to five times. Recoveries were between 78% and 95%, and relative standard deviations of reproducibility were between 6.6% and 13.1%, depending on concentration level. The determination limit was 0.05 μg/g.
Hormone analysis.
Testosterone, progesterone, and luteinizing hormone (LH) were analyzed in serum from the pups at GD 21, PND 16, and PND 224. Testosterone and progesterone were extracted from the serum as previously described (Vinggaard et al., 2002; Birkhj et al., 2004) and the hormones were measured by time-resolved fluorescence using commercially available fluoroimmunoassay (FIA) kits (PerkinElmer Life Sciences, Turku, Finland).
Luteinizing hormone was analyzed at Turku University, Finland, using an immunofluorimetric assay as described by Haavisto et al. (1993).
Ex vivo testosterone and progesterone production at GD 21 was determined by decapsulating and incubating the testes in a shaking water bath at 34°C for 3 h in DMEM/F12 medium containing 0.1% BSA. Vials were centrifuged at 4000 x g for 10 min and supernatants were stored at –20°C until hormone levels were analyzed by the above-mentioned methods.
Cytochrome P450 activity analysis.
The ethoxyresorufin-O-deethylase activity (EROD) was measured in liver microsomes from PND 16 males and females using black microtiter plates (Black Optiplate, Packard). The reaction mixture contained 6 μg of liver microsomal protein and 0.1 M Tris–HCl (pH 7.8) containing 1.6 mg/ml BSA and 2 μM ethoxyresorufin (ER) in a total volume of 0.2 ml. The mixture was pre-incubated at 37°C for 2 min, and the reaction was initiated by the addition of 0.25 mM NADPH. Resorufin-associated fluorescence was measured using a fluorescence reader (Victor2 Wallac, PerkinElmer, Life Sciences, Denmark) employing an emission wavelength of 590 nm and a 560-nm excitation wavelength. The reaction was followed for 30 min with readings every 2 min, and the dealkylation rate was estimated on the basis of a resorufin standard curve ranging from 0 to 400 nM. Each sample was measured in duplicate and reaction rates were calculated. Benzyloxy- (BROD), methoxy- (MROD) and pentoxyresorufin-O-deethylase (PROD) activities were measured following the same procedures as described for the EROD activity.
It should be kept in mind that the dealkylation processes are only surrogate markers for CYP1A1, CYP1A2, CYP2B, and CYP3A, respectively. The authors are aware that some of the alkylresorufin substrates can be metabolized by several CYP isozymes, however, only the predominant CYP enzymes involved in the dealkylation reactions are listed above (Burke et al., 1994).
Section of adult males and semen quality analysis.
At PND 224 (32 weeks) adult males and females were killed and the same organs were taken out from the males as for PND 16 animals. The right testis was fixed in Bouin's fixative for histopathology, and the left testis was used for testosterone measurement.
Cauda of the right epididymis was used for motility analysis. Spermatozoa were collected by a modified Poke method (Slott and Perrea, 1993) and contained in Medium 199 Hanks & Hepes (Invitrogen Life Technologies, DK), supplemented with 0.5% bovine serum albumin (Sigma Chemical Company, St. Louis, MO). Sperm samples were analyzed by computer-assisted sperm analysis (CASA; HTM-IVOS version 12 Hamilton Thorne Research, Beverly, MA). The parameters evaluated were percent motile sperm and sperm velocities. Cauda of the left epididymis was frozen in liquid nitrogen for later sperm count. Cauda was thawed at room temperature, trimmed of fat, weighed, and homogenized in 1 ml Triton X-100. The homogenate was treated with ultrasonic sound and placed into a stain reaction vial containing DNA-specific fluorescent stain (Supra Vital IDENT Stain Kit, HTR). Sperm samples were placed in the HTM-IVOS. Samples were analyzed by CASA, counts were averaged, and the data were presented as number of sperm per gram cauda.
Behavioral studies.
The investigations were performed between 8.00 A.M. and 3 P.M. during the last of hour of the light cycle and during the dark cycle of the animals, i.e., their active period. The experimenter was kept unaware of the group to which an individual rat belonged. Exposed and control animals were tested alternately as were female and male animals. The ages for the assessment of end points were carefully planned and generally reflect that the same offspring were evaluated for all end points. For several end points, such as motor activity, Morris water maze learning, and sweet preference, the animals have to be sexually mature in order to find sexual dimorphic behavioral differences. In addition, the sweet preference test and the radial arm maze require single housing of the animals, and so these were the last tests to be performed.
Motor activity and habituation capability.
The activity of the animals was recorded in activity boxes with photocells for 10 x 3 min at PND 22 ± 1 prior to weaning, at PND 28 ± 1, and in adult animals at the age of 12 weeks. The total activity during the 30 min was used as a measure of general activity. In order to assess habituation capability, the 30 min was divided into two time periods of 15 min.
Play behavior.
The play behavior test was performed when the offspring were 34 ± 1 days old. The day before the test, the animals were housed individually in clean cages. Immediately before testing, the animals were placed in pairs with their usual littermate in their normal cage, and their behavior was recorded on videotape for 10 min. Afterwards, the latency to initiate play behavior, and the number of pinnings was scored. The test was performed in a room with no light, and behavior was recorded with a photosensitive camera.
Learning and memory (Morris water maze).
The animals were tested at the age of 13–14 and 17–18 weeks in a Morris water maze with a diameter of 220 cm as described earlier (Hass et al., 1995), with minor modifications. Four points on the rim of the pool, N, E, S, and W (not true magnetic directions), were used as starting points. A circular transparent platform was situated 1 cm below the water surface, and thus was invisible from water level. The animals were tested in four daily trials using the four starting points assigned in a pseudo-random sequence. The scheme used was: initial learning during 5 days; memory after 4 weeks for 2 days; reversal learning with the platform opposite the original location for 1 day; and new learning with the platform in the center for 1 day. A video-tracking device (Viewpoint video tracking system, Sandown Scientific, Middlesex, England) tracked the route of the animals; the latencies to find the platform, the path lengths, and swimming speeds were used as end points, as described in Hass et al. (1995).
Radial arm maze.
At the age of 22–27 weeks, the animals were investigated in a standard 8-arm radial arm maze as described elsewhere (Lam et al., 1991). One week before testing, the animals were housed one per cage and given restricted access to food in the afternoon (approximately 30 min per day). Once a day, they were given a few small pieces of the reward (peanut chips) to be used during testing in the radial arm maze. The food restriction continued throughout the testing period. The animals were tested daily in a total of 15 sessions over 3 consecutive weeks (5 trials per week). All eight arms in the maze were baited with peanut chips. In the daily test session, each rat was placed in the central maze area and allowed to explore the maze either until all chips were collected or until 10 minutes had elapsed. Latency to pass all the distally placed photoelectric cells and choice of arms was registered by computer. The number of errors, defined as visiting an arm that had already been visited, was calculated from the data.
Sweet preference test.
At the age of 21 weeks the animals were investigated in the sweet preference test. The week before testing, the animals were accustomed to drinking from two bottles with normal water. During testing, the animals were given the choice between normal water and water sweetened with 0.25% saccharin for 3 days. To avoid bias from preference of the left or right bottle, the placement of the bottles was equally distributed in the groups. The volumes of the solutions were determined each day. Body weight was registered in the same week and used for calculation of saccharin intake per 100 g body weight.
Statistical Analyses
Statistical evaluation of plasma hormone data, CYP 450 data, and semen data.
A one-way analysis of variance (ANOVA) was employed for all groups and, if significant, was followed by the post hoc test, Dunnett's test. Significance was judged at p < 0.05.
Sperm data were examined for normal distribution and homogeneity of variance. Single animal data were analyzed in a one-way ANOVA (proc glm) followed by Dunnett's t-test (version 8, SAS Institute Inc, Cary, NC).
Statistical evaluation of hormone levels and organ weights.
For statistical evaluation of testosterone and progesterone levels in testes (2–4 males per litter) and ex vivo testosterone and progesterone production at GD 21 (3–11 males per litter), all males were included in the analysis. Data from one male and one female per litter at PND 16 (12–16 litters per group) and one or two males per litter at PND 224 (11–16 litters per group leading to a total of 16 animals per group) were used to analyze terminal body weight and organ weights. To adjust for litter effects, litter was included in the ANOVA as an independent, random, and nested factor (proc mixed, SAS version 8, SAS Institute). Organ weights were analyzed using treatment as one main factor and age as another main factor, and body weight was used as a covariate. Non-processed and ln-transformed data were examined for normal distribution and homogeneity of variance. If an interaction between age groups and dose group was observed, the age groups were analyzed separately. When an overall significant treatment effect was observed, two-tailed comparison was performed using least squares means. In cases where normal distribution and homogeneity of variance were not obtained, data were additionally tested with the non-parametric Wilcoxon Scores, followed by a Kruskall-Wallis test. Effects on testis histopathology were analyzed by Fisher's exact test. A level of p < 0.05 was considered as statistically significant.
Statistical evaluation of pregnancy data, litter data, and behavioral data.
The litter was generally considered the statistical unit and the alpha level was 0.05. When more than one pup per litter is investigated, two closely related statistical strategies are available to avoid inflation of sample size (Holson and Pearce, 1992). The first alternative uses one score per litter, either a litter mean or the score of a single animal per litter. Second, and theoretically most attractive, one can include litter as an independent, random, and nested factor in the ANOVA. This approach specifically controls for litter effects and offers a direct statistical test of the significance of such effects. The two approaches are mathematically identical in their test for treatment effects (Holson and Pearce, 1992).
Data from the pre-weaning period were analyzed using litter means. Analyses of co-variance (ANCOVA) were used to test the differences between the groups in birth weights and pre-weaning pup weights while controlling for litter size. The remaining results were analyzed by ANOVA, when relevant, with nested design and repeated measures in trials or days. The SYSTAT PC-version software package was used for all computations (Systat 1990).
RESULTS
Pregnancy and Litter Data
Prochloraz had no effects on maternal weight gain during the period from GD 7 to GD 21 or on maternal weights on PND 1 (Table 1). However, a significant reduction in maternal weight gain from GD 7 to PND 1 was detected, which means that the prochloraz-dosed dams gained less weight than the control dams did. The mean pregnancy length was significantly increased by around 12 h after exposure to 30 mg/kg prochloraz. The control dams, as expected, all gave birth on GD 22 or GD 23 (6 and 10 dams, respectively). Among the dams exposed to prochloraz, none gave birth on GD 22; 15 on GD 23; and 1 on GD 24. Therefore, the mean increase in gestation length of 12 h reflects that birth is delayed by approximately 1 day for half of the animals. However, no effects on pup weight, sex ratio, litter size, postnatal death, or postimplantation-perinatal loss were detected. The pesticide mixture caused significantly reduced maternal weights after birth and decreased maternal weight gain from GD 7 to after birth. No effect on birth weight was found, but the body weights of the pups were significantly decreased at PND 6 and PND 13.
Chemical Analysis of Breast Milk
Chemical analysis of the content of prochloraz in rat's breast milk was performed by analyzing the milk content of stomachs from female pups sacrificed on PND 8/9. The results showed that no detectable levels were found in the control group (n = 6), whereas the concentration in the prochloraz and mixture group was measured to be 5.3 ± 3.9 μg/g (n = 13) and 1.5 ± 1.7 μg/g (n = 9), respectively.
On the basis of the pup weights at PND 0, 6, and 13 (Table 1), the weight gain of the pups was estimated to be 2 g/day during PNDs 6–9 and the body weight on PND 9 to be around 19 g. Assuming that the milk intake corresponds to approximately 2 ml/10 g body weight, the dose transferred at PND 9 to the pups via the breast milk can be estimated to be:
Thus, around 3% of the maternal dose of 30 mg/kg/day seems to be transferred to the pups on PND 9.
Anogenital Distance and Nipple Retention
Neither prochloraz alone nor the mixture affected anogenital distance in the male offspring at the selected dose levels (Table 1). A statistically significant effect on nipple retention at PND 13 was seen in the pups exposed to prochloraz, and the percentage of litters with nipples in male pups was significantly increased in both the prochloraz and the mixture group (Fig. 3). In the male fetuses at GD 21, no nipples or areolas were observed.
Hormone Levels GD 21, PND 16, and PND 224
Plasma testosterone, testicular testosterone and progesterone, and plasma LH were analyzed in GD 21 fetuses taken by cesarean section. The data showed that both prochloraz and the mixture significantly reduced plasma and testicular testosterone, whereas the testicular progesterone level was increased (Fig. 2). There was a slight tendency toward increased plasma LH levels, although this was not statistically significant. In PND 16 males serum testosterone, progesterone, and LH were not significantly affected, and in adult males (PND 224) no effects on testicular testosterone levels were found.
Basal testosterone and progesterone production ex vivo was investigated at GD 21 (Fig. 2). The reduced testosterone synthesis was statistically significant for the mixture group, but not for the prochloraz-dosed animals, whereas progesterone synthesis was significantly increased for both the prochloraz group and the mixture group.
Development of Pups (Organ Weights, Histology, and Sexual Maturation)
Body and organ weights for PND 16 males and females are shown in Table 2. The only significant effects that were detected were a decreased weight of bulbourethral glands caused by prochloraz, and a reduced body weight of males and females exposed to the mixture. The effect on body weight was also evident at PND 22, but only in the females at sexual maturation (Table 3). The day of sexual maturation as determined by vaginal opening and prepuce separation was unaffected in both male and female offspring. At PND 224 no effects on organ weights were seen for either prochloraz or the mixture (data not shown).
The testes were investigated macroscopically and microscopically by a blinded approach on GD 21, PND 16, and PND 224. At GD 21 moderate Leydig cell hyperplasia was observed in one of 10 males in the control group, in 6 of 18 males in the prochloraz group, and in 2 of 10 males in the mixture group. In a Fisher's exact test the increased incidence of Leydig cell hyperplasia was not statistically significant. At PND 16 and PND 224 no obvious treatment-induced histopathological effects were observed in the testis.
Cytochrome P450 Activities
The effect of prochloraz and the mixture on selected drug-metabolizing enzymes was investigated in livers of PND 16 males and females. From Figure 4 it is evident that prochloraz produced a minor induction of hepatic ethoxyresorufin O-dealkylase (EROD) and methoxyresorufin O-dealkylase (MROD) activities in both males and females, whereas neither penthoxyresorufin O-dealkylase (PROD) nor benzyloxyresorufin O-dealkylase (BROD) were affected. The mixture only significantly affected EROD and MROD in the females.
Semen Quality
Sperm counts were analyzed in PND 224 males. In the control, prochloraz, and mixture groups the sperm numbers were found to be 412 x 106 ± 68 (n = 16), 383 x 106 ± 60 (n = 16), and 375 x 106 ± 60 (n = 16), and no statistically significant effect was found. No effects on number of motile sperm cells or sperm velocities were detected either.
Behavioral Studies
Investigations of animal behavior comprised tests for play behavior, memory and learning, motor activity, and sweet preference. No exposure-related differences were found on play behavior or learning and memory functions, but the expected gender-related differences in behavior were found (data not shown). The motor activity was not affected in the prepubertal offspring, and no gender differences were seen. The latter finding was expected because gender differences in motor activity levels are usually not seen until after puberty (Blizard et al., 1975). The motor activity levels of the adult males and females were, as expected, statistically significantly different, with females exhibiting higher activity level than males. In the prochloraz-exposed adult males, a statistically significantly increased level of motor activity was found (Fig. 5). There was also a tendency toward a similar effect in the males exposed to the mixture, but the difference was not statistically significant (p = 0.064). The investigation of sweet preference showed, as expected, a clear sex difference, with females exhibiting higher preference for sweetened water than males. The males exposed to prochloraz generally showed a higher preference than control males during the 3 days of the test, but the differences were only statistically significant on the second day (Fig. 6).
DISCUSSION
Prochloraz was found to cause a small but statistically significant increase in mean pregnancy length of approximately 12 h. This result reflects that approximately half of the prochloraz animals gave birth 1 day later than expected. A similar effect was found by Noriega et al. (2003) after administration of higher doses of 62.5, 125, 250, or 500 mg/kg prochloraz to pregnant dams GD 14–18. In some high-dose–treated dams in that study, pup delivery was delayed up to 30 h. Previously, a classical 2-generation study with prochloraz was performed in rats (WHO, 1983). Prochloraz was given in the diet at 0, 37.5, 150, or 650 ppm (31 mg/kg) prior to mating and throughout mating, gestation, and lactation. An extended gestation period was also seen in this study, together with reduced food consumption, whereas no significant effects on mating performance or pregnancy rates were found.
The marked prochloraz-induced reduction in testosterone level and increase in progesterone level at GD 21 were both found in testis and plasma. In agreement with this finding, ex vivo progesterone production was significantly increased after prochloraz administration. However, no statistically significant effect on ex vivo testosterone production was found after administration of prochloraz 30 mg/kg alone. Wilson et al. (2004) have reported that, after administration of 250 mg/kg prochloraz between GD 14 and GD18, a reduced ex vivo testosterone production at GD 18 by around 50% was found, together with an increase in progesterone production (7.5-fold). In our study a dose of 30 mg/kg prochloraz was able to reduce fetal testosterone synthesis and increase fetal progesterone synthesis, indicating that the steroidogenic synthesis pathway in Leydig cells is directly affected by prochloraz. These changes in hormone levels are reversible, as we found no changes in testosterone and progesterone levels at PND 16 and no change in testosterone at PND 224.
It is conceivable that the delayed delivery of the dams, the increased progesterone levels, and the decreased testosterone levels in the fetuses are interconnected in some way. The increase of progesterone and the decrease of testosterone may be caused by an inhibition of 17-hydroxylase/17,20-lyase, 3-hydroxysteroiddehydrogenase and/or 17-hydroxysteroiddehydrogenase. As imidazole compounds have been shown to inhibit 17-hydroxylase and 17,20-lyase but not to have marked effects on the other two enzymes (Ayub and Levell, 1987), it is conceivable that this mechanism of action has played a major role. In the female rat the regression of the corpus luteum and a decline of progesterone secreted by corpus luteum in high amounts during pregnancy is a prerequisite for initiation and facilitation of delivery (Christenson and Deveto, 2003). Thus, although progesterone levels were not measured in the dams, the increased gestational length in prochloraz-dosed animals may be due to impaired luteolysis and increased progesterone levels.
A high plasma progesterone level in dams, leading to delayed parturition in rats, has been observed for fenarimol (WHO, 1995), a fungicide that resembles prochloraz in that it is both an aromatase inhibitor and an AR antagonist (Vinggaard et al., 2000, 2005).
Nipple retention was found in male pups exposed to prochloraz and, to a lesser extent, in pups exposed to the mixture. As the dose of prochloraz alone was twice as high as the dose given in the mixture, these findings indicate a dose–response relationship of prochloraz. No effects were found on the anogenital distance (AGD) of the male pups at birth. Both AGD and nipple retention are normally affected after prenatal exposure to antiandrogens (Gray et al., 2001). The lack of effect on AGD, together with a clear effect on nipple retention in the present study, indicates that nipple retention is a more sensitive end point than AGD after prochloraz exposure. The fact that increased nipple retention was also found by Noriega et al. (2003) after exposure GD 14–18 indicates that the underlying cause of this adverse effect involves factors taking place during gestation. Gray et al. (1999a) have described antiandrogens by categorizing several chemicals according to their pseudohermaphroditism index (PHI). One PHI score was calculated for effects on DHT-dependent tissues (AGD, areolas, retained nipples, ventral prostate size, hypospadias, and vaginal pouching) and one was given for effects on testosterone-dependent effects (seminal vesicle, epididymal, and testicular size or percent abnormal). The AR antagonists vinclozolin, procymidone, and p,p'-DDE all have the most pronounced effects on DHT-dependent tissues, whereas the phthalates that inhibit fetal steroidogenesis have the most marked effects on testosterone-dependent tissues. In the present study prochloraz exposure caused increased nipple retention and decreased bulbourethral gland weight, and we know that at higher doses the anogenital distance is reduced (Noriega et al. (2003) and own unpublished results). Because dihydrotestosterone (DHT) has been shown to be the proximal androgen for growth of the bulbourethral glands in mouse organ cultures (Cooke et al., 1987), these results indicate that DHT-dependent tissues are more severely affected by prochloraz. Although AR antagonism may be involved, the present data suggest that testosterone biosynthesis at the level of CYP 450 hydroxylase/lyase may mediate the observed DHT-mediated tissue alterations.
Prochloraz was found to induce the activity of CYP1A enzyme activities (EROD and MROD), but not BROD or PROD activities in PND 16 animals. This finding is in agreement with our previous result that prochloraz is an agonist of the Ah receptor (Long et al., 2003), leading to induction of CYP1A activities, whereas CYP2B activities were unaffected. Prochloraz has previously been reported to increase liver weights after administration of doses of 25 mg/kg and higher in castrated testosterone-treated rats (Birkhj et al., 2004; Vinggaard et al., 2002). This effect on liver weight may be a result of hypertrophy of liver cells, which may be caused by induction of CYP 450 activities. A previous study in which 250 mg/kg prochloraz was orally dosed to adult male rats for 3 days showed that both EROD (22-fold) and PROD (2.4 fold) were induced, which led to the suggestion that prochloraz is a mixed inducer of CYP 450 (Laignelet et al., 1989). In the present study we did not observe any effects on PROD, which we believe is due to the differences in dosages used in the two studies (30 mg/kg versus 250 mg/kg). It may be speculated that part of the antiandrogenic effect of prochloraz could be due to increased metabolism of testosterone via CYP 450 induction. However, as progesterone was increased, this seems not to be a plausible hypothesis.
In the present study no severe malformations of the reproductive system were observed. However, using this study design, in which animals were autopsied at various ages, may have decreased the power of the study compared to a design, in which all males are kept until adulthood and investigated for malformations, like the study reported by Foster and McIntyre (2002).
It is well known that there is a natural sex difference in the reproductive behavior of male and female rats. In addition, sex differences in a large number of other behavioral end points depend on early gonadal hormone secretion (MacLusky and Naftolin, 1981). Sex differences in central nervous function represent the outcome of interactions between several different factors, among which the hormones secreted by the gonads during development are of paramount importance. In mammals, the intrinsic behavioral pattern is generally female, with differentiation toward the masculine pattern of behavior occurring in the male as a result of exposure to testicular hormones during development. The conversion of testosterone into estradiol by aromatase in the brain, however, is critical for the organization of the male brain in rats (Hotchkiss et al., 2002). It has been shown in animal models that adult female rats have a higher spontaneous activity behavior than males and that they have a higher preference for sweetened water in a "sweet preference test" (MacLusky and Naftolin, 1981; Valenstein et al., 1967). Gender differences for spatial learning abilities have been reported in both humans and rodents, where males generally perform better than females (Roof, 1993). These gender-related differences were confirmed in the present study, including the activity and the sweet preference test. In these tests, we found that the prochloraz-exposed males in adulthood exhibited behavioral effects pointing toward incomplete masculinization/feminization of their behavior. It is conceivable that the feminized behavior of the male rats receiving prochloraz is a consequence of the markedly reduced testosterone levels found in this study during the late gestation period. Prochloraz has, however, also been shown to inhibit aromatase activity (Vinggaard et al., 2000), and this may have contributed to the effect by inhibiting the conversion of testosterone into estradiol, thereby reducing the estradiol levels needed for complete behavioral masculinization of the male rat brain.
In general, qualitatively similar, but apparently weaker effects were seen after exposure to the mixture compared to prochloraz administration alone. This was true for effects on hormone levels on GD 21, nipple retention, CYP 450 activities, and animal behavior. We believe that these effects of the mixture are due to the presence of 15 mg/kg prochloraz, which should be compared to the single dose of 30 mg/kg given in the prochloraz group. There are only few indications of differential effects between prochloraz and the mixture. These are the slight maternal toxicity of the mixture as seen from the reduced maternal weight on PND 1 and reduced weight of the pups after (but not before or at) birth, which suggests that pup growth is more vulnerable to the mixture during lactation than during gestation. Furthermore, a statistically significant reduction of the ex vivo testosterone production was seen for the mixture group, but not for the prochloraz group, and it may be speculated that the mixture component simazine, which has been reported to be an aromatase inducer (Sanderson et al., 2000), might have contributed to the lowered fetal testosterone levels. Overall, the results indicate that the relatively low doses of deltamethrin, methiocarb, simazine, and tribenuron-methyl present in the mixture did not markedly affect the response to prochloraz. The results further indicate that the non-AR active compounds (simazine and tribenuron-methyl) were not able to markedly modify the response caused by the AR-blocking compounds (deltamethrin, methiocarb, and prochloraz).
In conclusion, we have shown that prochloraz, apart from being able to block AR, is able to affect fetal steroidogenesis, two potential mechanisms of action for its antiandrogenic effects. These effects are suggested to be the cause of the increased nipple retention, the reduced weight of bulbourethral glands, and the feminized behavior of the male offspring. The adverse effects are subtle, but they occur after administration of a relatively low maternal dose of 30 mg/kg, and it was estimated that around 3% of this dose was transferred to the pups. It is of concern that a fungicide with these effects has been registered for use in many countries, and consideration should be given to reducing the risk of human exposure to this fungicide.
ACKNOWLEDGMENTS
This study was supported by the Danish Environmental Protection Agency and the Danish Research Council (grants no. 9801270 and 22–03–0198). We are indebted to Birgitte Mller Plesning, Rico Wellendorph Lehmann, Lisbeth Tranholm Andersen, Morten Andreasen, Lillian Sztuk, Bo Herbst, Ulla El-Baroudy, and Trine Gejsing for excellent technical assistance.
REFERENCES
Andersen, H. R., Vinggaard, A. M., Rasmussen, T. H., Gjermandsen, I. M., and Bonefeld- Jorgensen, E. C. (2002). Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicol. Appl. Pharmacol. 179, 1–12.
Ayub, M., and Levell, M. J. (1987). Inhibition of testicular 17-hydroxylase and 17,20-lyase but not 3-hydroxysteroid dehydrogenase-isomerase or 17-hydroxysteroid oxidoreductase by ketoconazole and other imidazole drugs. J. Steroid Biochem. 28, 521–531.
Birkhj, M., Nellemann, C., Jarfelt, K., Jacobsen, H., Andersen, H. R., and Vinggaard, A. M. (2004). The combined antiandrogenic effects of five commonly used pesticides. Toxicol. Appl. Pharmacol. 201, 10–20.
Blizard, D. A., Lippman, H. R., and Chen, J. J. (1975). Sex differences in open-field behavior in the rat: The inductive and activational role of gonadal hormones. Physiol. Behav. 14, 601–608.
Burke, M. D., Thompson, S., Weaver, R. J., Wolf, C. R., and Mayer, R. T. (1994). Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem. Pharmacol. 48, 923–936.
Christenson, L. K., and Devoto, L. (2003). Cholesterol transport and steroidogenesis by the corpus luteum. Reprod. Biol. Endocrinol. 1, 90.
Cooke, P. S., Young, P. F., and Cunha, G. R. (1987). A new model system for studying androgen-induced growth and morphogenesis in vitro: The bulbourethral gland. Endocrinology 121, 2161–2170.
Foster, P. M., and McIntyre, B. S. (2002). Endocrine active agents: Implications of adverse and non-adverse changes. Toxicol. Pathol. 30, 59–65.
Gray, L. E., Jr., Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R. L., and Ostby, J. (1999a). Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol. Ind. Health 15, 94–118.
Gray, L. E., Jr., Ostby, J., Monosson, E., and Kelce, W. R. (1999b). Environmental antiandrogens: Low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol. Ind. Health 15, 48–64.
Gray, L. E., Jr., Ostby, J., Furr, J., Wolf, C. J., Lambright, C., Parks, L., Veeramachaneni, D. N., Wilson, V., Price, M., Hotchkiss, A. et al. (2001). Effects of environmental antiandrogens on reproductive development in experimental animals. Hum. Reprod. Update 7, 248–264.
Haavisto, A. M., Pettersson, K., Bergendahl, M., Perheentupa, A., Roser, J. F., and Huhtaniemi, I. (1993). A supersensitive immunofluorometric assay for rat luteinizing hormone. Endocrinology 132, 1687–1691.
Hass, U., Lund, S. P., Simonsen, L., and Fries, A. S. (1995). Effects of prenatal exposure to xylene on postnatal development and behavior in rats. Neurotoxicol. Teratol. 17, 341–349.
Henry, M. J., and Sisler, H. D. (1984). Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi. Pesticide Biochem. Physiol. 22, 262–275.
Holson, R. R., and Pearce, B. (1992). Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species. Neurotoxicol. Teratol. 14, 221–228.
Hotchkiss, A. K., Ostby, J. S., Vandenbergh, J. G., and Gray, L. E., Jr. (2002). Androgens and environmental antiandrogens affect reproductive development and play behavior in the Sprague-Dawley rat. Environ. Health Perspect. 110, 435–439.
Korenbrot, C. C., Huhtaniemi, I. T., and Weiner, R. I. (1977). Preputial separation as an external sign of pubertal development in the male rat. Biol. Reprod. 17, 298–303.
Laignelet, L., Narbonne, J.-F., Lhuguenot, J.-C., and Riviere, J.-L. (1989). Induction and inhibition of rat liver cytochrome(s) P-450 by an imidazole fungicide (prochloraz). Toxicology 59, 271–284.
Laignelet, L., Rivière, J.-L., and Lhuguenot, J.-C. (1992). Metabolism of an imidazole fungicide (prochloraz) in the rat after oral administration. Food Chem. Toxicol. 30, 575–583.
Lam, H. R., Larsen, J.-J., Ladefoged, O., Mller A., Strange P., and Arlien-Sborg, P. (1991). Effects of 2,5-hexanedione alone and in combination with acetone on radial arm maze behavior, the "brain-swelling" reaction and synaptosomal functions. Neurotoxicol. Teratol. 13, 407–412.
Long, M., Laier, P., Vinggaard, A. M., Andersen, H. R., Lynggaard, J., and Bonefeld-Jrgensen, E. C. (2003). Effect of currently used pesticides in the AhR-CALUX assay: Comparison between the human TV101L and the rat H4IIE cell line. Toxicology 194, 77–93.
Maclusky, N. J., and Naftolin, F. (1981). Sexual differentiation of the central nervous system. Science 211, 1294–1303.
Needham, D, Creedy, C. L., and Dawson, J. R. (1992). The profile of rat liver enzyme induction produced by prochloraz and its major metabolites. Xenobiotica 22, 283–291.
Noriega, N. C., Gray, L. E., Jr., Ostby, J., Lambright, C., and Wilson, V. S. (2003). Prenatal exposure to the fungicide prochloraz alters the onset of parturition in the dam and sexual differentiation in male rat offspring. Toxicologist 72 (S1), Abstract 1375, p.283.
Poulsen, M.E., and Granby, K. (2000). Validation of a multiresidue method for analysis of pesticides in fruit, vegetables and cereals by GC/MS iontrap system. In Principles and Practices of Method Validation (A. Fajgelj and A. Ambrus, Eds.), Special Publication No 256 from The Royal Society of Chemistry, Cambridge, UK. ISBN 0–85404–783–2.
Roof, R. L. (1993). Neonatal exogenous testosterone modifies sex differences in radial arm and Morris water maze performance in prepubescent and adult rats. Behav. Brain Res. 53, 1–10.
Sanderson, J. T., Seinen, W., Giesy, J. P., and van den, Berg M. (2000). 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: A novel mechanism for estrogenicity Toxicol. Sci. 54, 121–127.
Slott, V. L., and Perrea, S. D. (1993). computer-assisted sperm analysis of rodent epididymal sperm motility using the Hamilton-Thorn Motility Analyzer. In Methods in Toxicology, Part A, Male Reproductive Toxicology (Chapin, R. E., and Heindel, J. J., Eds.), vol. 3, chapter 20, Academic Press, Inc., San Diego, CA.
Swan, S. H., Kruse, R. L., Liu, F., Barr, D. B., Drobnis, E. Z., Redmon, J. B., Wang, C., Brazil, C., and Overstreet, J. W. (2003). Semen quality in relation to biomarkers of pesticide exposure. Environ. Health Perspect. 111, 1478–1484.
Valenstein, E. S., Kakolewski, J., and Cox, V. C. (1967). Sex differences in taste preference for glucose and saccharin solutions. Science 1565, 942–943.
Vinggaard, A. M., Hnida, C., Breinholt, V., and Larsen, J. C. (2000). Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol. in Vitro 14, 227–234.
Vinggaard, A. M., Nellemann, C., Dalgaard, M., Jorgensen, E. B., and Andersen, H. R. (2002). Antiandrogenic effects in vitro and in vivo of the fungicide prochloraz. Toxicol. Sci. 69, 344–353.
Vinggaard, A. M., Jacobsen H., Metzdorff, S. B., Andersen, H. R., and Nellemann C. (2005). Antiandrogenic effects in short-term in vivo studies of the fungicide fenarimol. Toxicology 207, 21–34.
Weidner, I. S., Moller, H., Jensen, T. K., and Skakkebaek, N. E. (1998). Cryptorchidism and hypospadias in sons of gardeners and farmers. Environ. Health Perspect. 106, 793–796.
WHO, 1983, Pesticide Residues in Food—1983 Evaluations. Part II—Toxicology, p. 366–442. WHO/PCS/61, International Programme on Chemical Safety, World Health Organisation, Geneva.
WHO, 1995, Pesticide Residues in Food—1995 Evaluations. Part II—Toxicology, p. 75–104. WHO/PCS/96.48, International Programme on Chemical Safety, World Health Organisation, Geneva.
Wilson, V. S., Lambright, C., Furr, J., Ostby, J., Wood, C., Held, G., and Gray, L. E., Jr., (2004). Phthalate ester–induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis. Toxicol. Lett. 146, 207–215.(Anne Marie Vinggaard, Sof)