ToxSci Advance Access originally published online on September 4, 2007
Toxicological Sciences 2007 100(2):464-473; doi:10.1093/toxsci/kfm227
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Endocrine-Disrupting Activities In Vivo of the Fungicides Tebuconazole and Epoxiconazole
* Department of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark
Department of Environmental Medicine, University of Southern Denmark, Winsløwparken 17, DK-5000 Odense C, Denmark
1 To whom correspondence should be addressed. Fax: +45-72-34-76-98. E-mail: ulh{at}food.dtu.dk.
Received July 6, 2007; accepted August 22, 2007
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ABSTRACT |
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The triazole fungicides tebuconazole and epoxiconazole were investigated for reproductive toxic effects after exposure during gestation and lactation. Rats were dosed with epoxiconazole (15 or 50 mg/kg bw/day) or tebuconazole (50 or 100 mg/kg bw/day) during pregnancy from gestational day (GD) 7 and continued during lactation until postnatal day (PND) 16. Some dams were randomly chosen for cesarean section at GD 21 to evaluate effects on sexual differentiation in the fetuses. Other dams delivered normally, and the pups were examined (e.g., anogenital distance [AGD] and hormone levels) at birth, at PND 13 or PND 16, and semen quality was assessed in adults. Both tebuconazole and epoxiconazole affected reproductive development in the offspring after exposure in utero. Both compounds virilized the female offspring as shown by an increased AGD PND 0. Furthermore, tebuconazole had a feminizing effect on male offspring as shown by increased nipple retention. This effect was likely caused by the reduced testosterone levels seen in male fetuses. Tebuconazole increased the testicular concentrations of progesterone and 17
-hydroxyprogesterone in male fetuses, indicating a direct impact on the steroid synthesis pathway in the Leydig cells. The high dose of epoxiconazole had marked fetotoxic effects, while the lower dose caused increased birth weights. The increased birth weights may be explained by a marked increase in testosterone levels in dams during gestation. Common features for azole fungicides are that they increase gestational length, virilize female pups, and affect steroid hormone levels in fetuses and/or dams. These effects strongly indicate that one major underlying mechanism for the endocrine-disrupting effects of azole fungicides is disturbance of key enzymes like CYP17 involved in the synthesis of steroid hormones. Key Words: tebuconaole; epoxiconazole; reproductive toxicity; virilization; fetotoxic.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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An endocrine disruptor (ED) is a synthetic chemical that when absorbed into the body either mimics or blocks hormones and disrupts the body's normal functions (http://www.nrdc.org/health/effects/qendoc.asp), and exposure to EDs during early life may cause long-term health effects (Murray et al., 2001
). The outcome of their action has been shown to have the potential to manifest itself in the embryo and fetus and also to have the ability to influence the development of offspring even until it reaches maturity or middle age (Colborn et al., 1993; Cooper and Kavlock, 1997; Murray et al., 2001). An increasing number of chemicals, including pesticides, are found to have endocrine-disrupting effects. Young children and fetuses are considered particularly vulnerable to pesticide exposure, due to a number of factors, including the fact that the nervous and reproductive organ systems are not fully developed until late childhood. While we have limited knowledge of the potential health risks, including endocrine effects, of low chronic doses of pesticides, some studies indicate an increased prevalence of cryptorchidism in sons of women working as gardeners (Weidner et al., 1998) or living on farms where pesticides have been used (Carbone et al., 2007; Kristensen et al., 1997).
Previously, we have tested a range of commonly used pesticides for their ability to disrupt the endocrine system using a battery of in vitro tests (Andersen et al., 2002; Long et al., 2003). The pesticides were chosen due to their frequent use in Danish green house horticultures. Two-thirds of the pesticides had endocrine-disrupting properties as evidenced by the significant response in one or more of the in vitro tests (aromatase activity, androgen-, estrogen- or aryl hydrocarbon receptor [AhR] assay). One of these pesticides was the imidazole fungicide prochloraz, which showed effect in all the in vitro tests that were run (Andersen et al., 2002; Long et al., 2003). Prochloraz proved to possess antiestrogenic and antiandrogenic effects to be an activator of the AhR and to inhibit the activity of the estrogen-synthesizing enzyme, aromatase. Subsequently, we found that prochloraz also induced antiandrogenic effects in rats in vivo in a Hershberger test as well as in a developmental toxicity study (Vinggaard et al., 2002; Vinggaard et al., 2005a). Furthermore, the antiandrogenic effect of prochloraz in a mixture containing four other pesticides was found to be additive (Birkhøj et al., 2004). This is supported by recent studies, in which results revealed that exposure to a mixture of antiandrogens with similar mechanisms of action (i.e., androgen receptor antagonism) can induce marked effects even when each chemical was present at doses associated with only weak, if any, effects (Hass et al., 2007; Metzdorff et al., 2007). Thus, it is important to bear in mind that even low exposures to EDs can contribute to a combined adverse effect, even though the effects of the single compounds are below the detection limit (Hass et al., 2007; Rajapakse et al., 2002; Silva et al., 2002).
The azole fungicides are used for the control of fungi in grain and to a lesser extent in flower, vegetable, and fruit production. In spite of the massive use of these pesticides, only few developmental toxicological studies of azole fungicides have been published. It is well-known from the literature that several among the investigated azole fungicides influence the activity of various cytochrome P450 enzymes, and one example is their inhibition of the activity of aromatase (CYP19) that converts androgens to estrogens (Mason et al., 1987; Sanderson et al., 2002; Vinggaard et al., 2000). One developmental toxicity study in vivo showed that exposure of pregnant rats to tebuconazole at dose levels around the lowest observed effect level (U.S. EPA, 1999) resulted in disturbance of the reproductive system in the offspring. The males had reduced weight of epididymides, and females had reduced uterus weight after peri- and postnatal exposure (Moser et al., 2001).
Our research on prochloraz, combined with the literature on other studies on azole fungicides, indicates that azole fungicides have the ability to act through multiple mechanisms and to induce various endocrine-disrupting effects (Vinggaard et al., 2005a,b).
The purpose of the present study was to investigate the endocrine-disrupting potential of two commonly used azole fungicides, epoxiconazole and tebuconazole, and to compare the results with the effects we have previously seen for prochloraz (Laier et al., 2006; Vinggaard et al., 2002; Vinggaard et al., 2005a). The chemical structures of epoxiconazole, prochloraz, and tebuconazole are illustrated in Figure 1. Compared to prochloraz, which is an imidazole, having the five-membered imidazole ring of three carbon atoms and two nitrogen atoms, epoxiconazole and tebuconazole are both triazoles, having a five-membered ring of two carbon atoms and three nitrogen atoms.
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The developmental effects of epoxiconazole and tebuconazole were investigated by dosing rats during pregnancy and further continued during lactation, followed by examination of the dams, the fetuses, or the newborn or young pups, as well as examination of semen quality in adult offspring.
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MATERIALS AND METHODS |
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Test Compounds
For the developmental toxicity study, we used epoxiconazole 99% pure (CAS no. 106325-08-8) and tebuconazole 98% pure (CAS no. 107534-96-3) from Dr Ehrenstorfer (Augsbrug, Germany). The vehicle was corn oil from Bie & Berntsen (Rødovre, Denmark).
Animals and Exposure
One hundred and twelve young adult time-mated Wistar rats (HanTac: WH, Taconic M&B, Ejby, Denmark) were supplied on gestational day (GD) 3. 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 (heat-treated) bedding (Tapvei, Gentofte, Denmark). 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 (9:00 P.M.–9:00 A.M.) and 12 h of darkness (9:00 A.M.–9:00 P.M.). The day after arrival, that is, GD 4, animals were weighed and assigned to five groups of 24, 20, 20, 24, and 24 animals, respectively, with similar weight distributions. An acclimatization period of 4 days was allowed before starting exposure. The rats were gavaged with 0, 50, or 100 mg/kg tebuconazole or 15 or 50 mg/kg epoxiconazole, respectively, from GD 7 to postnatal day (PND) 16. The doses were based on literature survey. Animals were divided into two sets, such that 56 animals representing all five groups were dosed 1 week prior to the next 56 animals. In the five groups, 19, 19, 18, 19, and 21 of the time-mated rats, respectively, were pregnant.
Health Status of Dams
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. 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 an effect on the maternal animal only.
Cesarean Sections GD 21
At the beginning of the study, 8 or 12 dams in each dose group were randomly selected for cesarean section. Of these the following were pregnant: six controls; seven dosed with 50 mg/kg, and eight dosed with 100 mg/kg tebuconazole (Teb); nine dosed with 15 mg/kg and 14 dosed with 50 mg/kg epoxiconazole (Epo). In the first set, additional cesarean sections on GD 24–25 had to be performed on two dams in the Teb-100 group and on four dams in the Epo-50 group, because the dams were unable to give birth and were diagnosed to have dystocia. Consequently, all Epo-50 dams in the second set of the study were selected for cesarean section on GD 21, giving a total group size of 14 pregnant dams in this group. The dams were weighed and decapitated after CO2/O2 anesthesia, uteri were taken out, and the number of live fetuses, location in uterus, resorptions, and implantations were registered. Body weight, sex, and any anomalies of the offspring were recorded. Trunk blood was collected immediately after decapitation into heparin-coated vials from all fetuses for hormone analysis, and one pool per litter was made for all male and female fetuses, respectively. Fetal testes were taken on GD 21 from one to three males per litter for determination of hormone levels or ex vivo testosterone production.
Delivery, Anogenital Distance, and Nipple Retention
The time of birth, weights of dams, and individual pups were recorded. The pups were counted, sexed, and checked for anomalies. Pups found dead were investigated macroscopically when possible. The day of delivery was designated PND 0. In all live pups, anogenital distance (AGD) was measured using a stereomicroscope. On PND 13, all male and female pups were weighed and examined for the presence of areolas/nipples, described as a dark focal area (with or without a nipple bud) located where nipples are normally present in female offspring.
Autopsy of Offspring on PND 16
The external genitals were inspected blinded to the observer at PND 16 in all males from all litters as described previously by Laier et al. (2006).
Organ Weight and Histopathology PND 16
From the control group, the Epo-15 and the Teb-50 groups one to three males and females from each litter were kept until adulthood for further studies. The rest of the pups were sacrificed on PND 16, and trunk blood was collected and within each litter pooled into a male and female sample. The pups were sacrificed on PND 16, as this was the last day of dosing of the dams. Consequently, the exposure of the pups through maternal milk will decrease, and it becomes difficult to assess the doses of ED that they are exposed to. Body weights of all male pups sacrificed on PND 16 were recorded. In one to two males per litter, the following organs were excised and weighed: liver, kidneys, adrenals, testes, epididymides, seminal vesicles, ventral prostate, bulbourethal glands, and the levator ani/bulbocavernosus muscles.
From one or two males per litter, the right or left testes were alternately fixed in Bouin's fixative, paraffin embedded, and stained with hematoxylin and eosin. In one male per litter, the following organs were fixed in formalin and embedded in paraffin: ventral prostate, seminal vesicles, epididymides, thyroids, and adrenals. All fixed organs were examined by light microscopy after staining with hematoxylin and eosin.
In one to two females per litter, body weights were recorded. The thyroid, ovaries, and uterus were excised and weighed from one female per litter.
Hormone Levels
Testosterone and progesterone, were analyzed in serum from the pups at GD 21 or PND 16 as described (Laier et al., 2006). Steroid hormones were analyzed in testis and estradiol in ovaries after extraction with diethyl ether. Testes or ovaries were placed in vials containing 100 or 500 µl water and 0.5 or 2.5 ml diethyl ether, respectively. The tissue was homogenized, and the vials were placed in a tub consisting of dry ice and acetone until the water fraction was frozen. The ether fraction was transferred to a clean vial, the procedure was repeated, and the two extracts were pooled and evaporated. Before analyzing, the samples were resuspended in 100 µl Diluent 1 (PerkinElmer, Turku, Finland) and incubated over night at 4°C. At the day of analysis, the samples were vortexed and incubated for 10 min at 45°C, before the hormones were measured by use of a Delfia time-resolved fluorescence kit (PerkinElmer) and measured by use of a Wallac Victor 1420 multilabel counter (PerkinElmer, Turku, Finland). Testicular 17-hydroxyprogesterone levels were analyzed by use of a 17-hydroxyprogesterone enzyme immunoassay kit from Assay Designs according to manufacturer's instructions (Electra-Box Diagnostica Aps, Denmark). Ex vivo testosterone and progesterone production at GD 21 was determined as previously described (Laier et al., 2006).
Semen Quality Analysis
Semen quality was analyzed in the controls and the low dose groups of tebuconazole and epoxiconazole. Due to fetal and neonatal toxicity in the highest dose groups, none of the high dose animals were kept for this analysis. The sexually mature males (7 months old) were anesthetized by CO2/O2 and decapitated. The epididymides were removed, and the cauda of the right epididymis was used for motility analysis. The cauda of the left epididymis was frozen in liquid nitrogen for later sperm count. A total of 11–12 animals per group were assessed, one to two males per litter.
Sperm motility.
Spermatozoa were obtained from the distal cauda, and sperm samples were prepared for analysis as previously described by Jarfelt et al. (2005). Sperm samples ware loaded into a 100-µm flat cannula (HTR 1099, Fercom, Denmark) and analyzed by computer-assisted sperm analysis; HTM-IVOS version 12 Hamilton Thorne Research, Beverly, MA). Minimum 20 fields were recorded at 60 Hz under x4 dark field illumination, and the images were video recorded for later analysis. The standard set up was used during analysis, and tracking errors were deleted through the edit and playback features. Twelve fields (minimum 200 sperm cells) were analyzed for each sperm sample. The samples were blinded to the observer. The parameters evaluated in this study were percent motile and percent progressive spermatozoa, curvilinear velocity, and amplitude of lateral head displacement that describe the vigor of the spermatozoa, and some progressive parameters such as average path velocity, straight line velocity, and straightness.
Sperm count.
Cauda of the left epididymis was thawed at room temperature and prepared for sperm count analysis as described by Jarfelt et al. (2005). Samples were placed in the HTM-IVOS and analyzed using 10 x UV fluorescent objective and IDENT OPTIONS set A. Ten fields were analyzed for each sample, and three counts were performed for each suspension. Counts were averaged, and data are presented as number of sperm per gram cauda.
Statistical Analyses
Data were examined for normal distribution and homogeneity of variance and, if relevant, transformed. In cases where normal distribution and homogeneity of variance could not be obtained by data transformation, a nonparametric Kruskall-Wallis test was used, followed by Wilcoxon's test for pairwise comparison. In all performed tests, statistical significance was judged at p < 0.05.
Statistical analysis of the effects on macroscopic lesions and histopathology were done using Fisher's Exact Test. For statistical evaluation of plasma hormone levels, a one-way ANOVA was employed for all groups and if significant, followed by a post hoc Dunnett's test.
When more than one pup from each litter was examined, statistical analyses was adjusted using litter as an independent, random, and nested factor in ANOVA. When an overall significant treatment effect was observed for the pregnancy and litter data, organ weights, and semen quality, a two-tailed comparison was performed using least square means. For the analysis of birth weight, AGD and nipple retention data from all offspring were analyzed. For statistical evaluation of testosterone and progesterone levels in testes, two to four males per litter were used, and for ex vivo testosterone and progesterone production on GD 21, 3–10 males per litter were used. Data analysis of AGD included the cubic root of body weight as a covariate in the analysis, to correct for the relationship between body size and AGD. In the analysis of organ weights PND 16, body weight was used as a covariate. Asterisks in tables and figures indicate a statistically significant difference compared to controls *p < 0.05, **p < 0.01. All analyses were done using SAS version 8, SAS Institute Inc., Cary, NC.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Developmental effects of epoxiconazole and tebuconazole were investigated by dosing pregnant dams during gestation or perinatally followed by examination of fetuses or offspring for effects on sexual differentiation.
Pregnancy and Litter Data
The high dose of tebuconazole decreased maternal weight gain during pregnancy, probably due to effects on both the dam and the uterine content (Table 1). Furthermore, the high dose of tebuconazole and epoxiconazole increased gestational length, loss of fetuses, and postnatal death of the pups, and the highest dose of epoxiconazole induced dystocia and a high frequency of stillbirth, leading to a marked reduction in live litter size. Many of the dead fetuses (27 of 128) had died very late in the gestation period, while such late fetal death was not seen in the controls (0 of 70). No significant toxic effects on fetuses or mothers were seen with the low dose of tebuconazole or epoxiconazole. Finally both tebuconazole and epoxiconazole caused decreased male and female fetal weight on GD 21 at the high dose.
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AGD and Nipple Retention
Exposure to either tebuconazole or epoxiconazole increased AGD in both female fetuses at GD 21 and in newborn female offspring (Fig. 2 and Table 1). In the male fetuses, significantly increased AGD was seen in both the tebuconazole and the epoxiconazole exposed fetuses, when dividing the AGDs with the cubic root of the body weight (Table 1). In the male offspring, no effects on AGD of tebuconazole or epoxiconazole were observed. However, AGD was significantly increased in the low epoxiconazole dose group before dividing this measure with the cubic root of the body weight (Table 1), but when AGD was divided by the cubic root of the body weight (Fig. 2), no changes was observed. Altogether there were indications of an effect on AGD in the males, but these effects were not consistent between fetuses and pups or between the AGD and the anogenital index.
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A statistically significant effect on nipple retention PND 13 was seen in the male pups exposed to tebuconazole. The increase in number of nipples was seen in both the low and high dose group of tebuconazole, and a similar trend—although not significant—was seen for epoxiconazole as well (Fig. 2 and Table 1).
Hormone Levels GD 21 or PND 16
Testicular testosterone, progesterone, and 17-hydroxyprogesterone were analyzed in male fetuses taken by cesarean section on GD 21. Testosterone levels were significantly decreased at GD 21 at the highest dose of tebuconazole. Both dose levels of tebucaonzole caused a significant increase in testicular 17-hydroxyprogesterone, as well as an increase in the progesterone levels, although only significant with the low dose. Epoxiconazole had no significant effect on the measured hormone levels in fetuses at GD 21 (Table 2). Neither compound had any effects on the ex vivo testicular testosterone and progesterone production examined at GD 21; however, a tendency toward an increase in the progesterone production was seen with the low dose of tebuconazole.
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In plasma from tebuconazole-dosed dams at GD 21, a marked increase in the progesterone level (sevenfold increase), as well as a significant decrease in T3 was seen. The high dose of epoxiconazole led to an increase in the progesterone level (sevenfold increase) as well as a twofold increase in the testosterone level (Table 3).
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As mentioned above, an increased AGD was seen in the female pups at birth (Fig. 2), and it was therefore of interest to analyze the estradiol levels in ovaries. A clear tendency toward lowered estradiol levels in female pups PND 16 was seen for both compounds; however, the effects were not statistically significant (Table 4).
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The plasma testosterone levels in males at PND 16 were unchanged, although a tendency toward a decrease was seen for the epoxiconazole-treated animals (Table 4).
Autopsy, Organ Weight, and Histopathology PND 16
Body and organ weights of male and female rat offspring at PND 16 are shown in Table 5. A statistically significant increased liver weight is observed at 100 mg/kg tebuconazole, and a tendency toward an increase in liver weight was seen after 50 mg/kg epoxiconazole as well. The high dose of epoxiconazole showed a tendency toward increasing the weights of all male reproductive organs (body weights unaffected), although this is questionable, due to the few animals available for the analysis. No effects on the reproductive organ weights or body weight were observed for either dose of tebuconazole.
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One testis from the low epoxiconazole dose group had no germ cells, but Sertoli cells only. Otherwise, no histopathological effects on the male reproductive organs were observed at PND 16 (data not shown).
Weights of female reproductive organs were unaffected by both compounds (Table 5).
Semen Quality Analysis
The number of sperm per gram cauda epididymis is shown in Figure 3. The group dosed with 15 mg/kg epoxiconazol had three males with severely reduced sperm number when compared to the control group (Fig. 3A), and furthermore, these three males had no motile sperm, as there were no sperm cells in the samples prepared for sperm motility analysis (Fig. 3B). Therefore, sperm quality data could not be analyzed by a parametric statistical procedure, and therefore a nonparametric test was run on all data. However, in order to investigate the effect on the remaining males, the severely affected dosed outliers were excluded from the statistical analyses, and an ANOVA was run on the remaining animals. No statistically significant reduction in the number of sperm cells was observed (Fig. 3A), and no statistically significant reductions were found when investigating any of the sperm motility and velocity parameters compared to the control group (Fig. 3B).
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The three affected animals from the epoxiconazole group had small, flaccid testes with a bluish color, and the testicular gubernacula adhered to the scrotum. Epididymides from these animals were smaller than normal and appeared to have a reduced content. Histologically, these testes were severely degenerated with areas of necrosis and fibrosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The main objective of this study was to investigate endocrine-disrupting effects of two frequently used triazole fungicides, epoxiconazole and tebuconazole, and compare the results with our previous findings on the imidazole fungicide, prochloraz. Prochloraz has been demonstrated to be antiandrogenic due to (1) androgen receptor antagonism in vitro; (2) a positive effect in the Hershberger assay, indicating either androgen receptor blocking and/or an effect on metabolism of testosterone; and (3) demasculinization of male pups after perinatal exposure in developmental toxicity studies in rats. In previous studies, it has been demonstrated that perinatal exposure to prochloraz caused feminization of male offspring and virilized female offspring (Laier et al., 2006
; Noriega et al., 2005; Vinggaard et al., 2005a). The results from the current study demonstrate that tebuconazole exposure also cause feminization of male offspring, as an increased number of nipples was observed at both doses (50 and 100 mg/kg bw/day), and the testosterone concentration in fetal testis at GD 21 was reduced in the highest dose group. Our conclusion is that the predominant effect of in utero tebuconazole exposure in male offspring is a feminization—an effect that resembles the effects induced by prochloraz (Vinggaard et al., 2005a). In female fetuses, both doses of tebuconazole caused increased AGD in fetuses, and for the highest dose, this effect was also evident at birth. This virilizing effect on female offspring is suggested to be a result of the increased progesterone levels in the dams (Willingham et al., 2006). Whether the tendency of decreased estradiol concentration in the ovaries at PND 16 plays a role as well is unclear. A virilizing effect on female offspring, after exposure to azole fungicides from GD 6 to PND 98, has also been documented by Rockett et al. (2006), where 2000 ppm myclobutanil in the feed, caused increased AGD in the female offspring at birth. The feminizing effect on males and virilizing effect on females are in agreement with another study demonstrating reduced weight of epididymides in adult male offspring and reduced uterus weight in adult female offspring after perinatal tebuconazole exposure (Moser et al., 2001), although the weight of epididymides and uterus were not affected in the offspring at PND 16 in our study. In the current study, the testicular progesterone and 17-hydroxyprogesterone levels in male fetuses were elevated after tebuconazole exposure and this, together with the lowered testosterone level, indicates a direct impact on the 17,20 lyase part of CYP17 in the Leydig cells. These effects on hormone concentrations and nipple retention for tebuconazole were comparable to our previous results for prochloraz (Vinggaard et al., 2005a).
Both AGD and nipple retention, which were measured in the current study, are 5-dihydrotestosterone (DHT)–dependent external effects (Gray et al., 1999). Since we are measuring testosterone and not DHT, a direct comparison between effects on AGD/nipples and testosterone can only be made, if it is assumed, that the chemicals tested do not affect 5-reductase activity.
Epoxiconazole acts differently in vivo than tebuconazole and prochloraz, as a marked fetotoxic effect was observed. The dams dosed with 50 mg/kg were in general unable to deliver their pups. Only two litters out of seven in the first set were born normally. Consequently, the litters in the second set were included in the cesarean sections at GD 21. Thus, the data for this dose of epoxiconazole on the live born pups are based on a very limited number of animals. In female offspring, AGD was increased in fetuses as well as in pups at birth indicating a virilizing effect. As for tebuconazole, the virilizing effect of epoxiconazole is supported by the increased progesterone levels in dams and perhaps by a tendency to lower estradiol concentration in the ovaries at PND 16. Our findings on epoxiconazole are in accordance with the studies reported, when epoxiconazole was approved for use in Denmark (EPA, 2003). In these studies, the fertility of male rats was reduced, gestational length was increased, the number of live born rat pups was reduced, and the weight of the adrenal glands in male offspring was reduced in a two-generation study in rats using doses between 30 ppm (approximately 3 mg/kg bw/day) and 1500 ppm (approximately 125 mg/kg bw/day) in the feed. No feminizing effects on male fetuses were seen with epoxiconazole in our study. In contrast, an increased birth weight was seen, which may be related to the marked up-regulated levels of testosterone in the dams. We suggest that the increased androgen exposure during pregnancy may have had a growth-promoting effect on the pups. Overall, epoxiconazole seems to alter sex hormone levels in the dams, but not in the fetuses.
Male offspring were feminized by tebuconazole but not by epoxiconazole. However, epoxiconazole was clearly fetotoxic, and this effect may mask any possible endocrine-disrupting effect in male pups. Both tebuconazole and epoxiconazole induced a high plasma concentration of progesterone in the dams. This is probably the reason for the increased gestational length as also previously suggested for prochloraz (Vinggaard et al., 2005a). In utero exposure to natural or synthetic progesterone can induce hypospadia in male mice, and the synthetic progesterone medroxyprogesterone acetate feminize male and virilizes female genitalia (Willingham et al., 2006). Thus, the high maternal progesterone concentration is likely to be involved in the virilizing effect on the female offspring.
Although mean reproductive organ weights and histopathology were unaffected by both tebuconazole and epoxiconazole at PND 16, abnormalities of testes and epididymides were observed in adulthood in 3 of the 12 animals from the epoxiconazole group. These males also had low sperm count and no motile sperm. Despite the low number of affected animals, this may be regarded as an effect of treatment with epoxiconazole, as it is in accordance with the low male fertility reported in the studies reviewed by the Danish Environmental Protection Agency (EPA, 2003).
The multiple mechanisms of action and complex patterns of endocrine-disrupting effects of azole fungicides are supported by a recently published study on reproductive toxicity of three triazole fungicides, myclobutanil, propiconazole, and triadimefon, administered in the feed from gestation through adulthood. Here it was found that the high doses of 2000, 2500, or 1800 ppm for myclobutanil, propiconazole, or triadimefon, respectively, increased AGD in male Wistar Han pups at PND 0, indicating hypervirilization. Increased maternal serum testosterone in the myclobutanil-dosed dams could account for the increased AGD of the male pups in this group, but no elevated serum testosterone levels from the dams in the propiconazole or triadimefon groups were reported (Goetz et al., 2007).
In conclusion, the results from our study reveal that not only prochloraz but also other azole fungicides have the abilities to act as EDs, although the profile of action in vivo varies. Tebuconazole possesses a more classical endocrine-disrupting effect on male pups like the one seen for prochloraz (i.e., increased nipple retention, reduced fetal testosterone, and increased progesterone). However, epoxiconazole is primarily fetotoxic, and secondly alters sex hormone levels in the dams, but not in the fetuses at the developmental stages assessed in this study. The common features between the two tested fungicides are the increased gestational length and the virilizing effect on female pups. Based on the present study, the conclusion is that both tebuconazole and epoxiconazole were capable of inducing effects on reproductive development in the offspring after exposure in utero. The effects on reproductive development after perinatal exposure strongly indicate that one of the main responsible mechanisms is disturbance of key enzymes involved in synthesis of steroid hormones. As both progesterone and 17-hydroxyprogesterone were elevated, it is possible that the lyase function of CYP17 is at least one of the targets for these azole fungicides. Recent data on the reproductive toxicity of triazole fungicides in male rats also point in the direction of disruption of testosterone homeostasis as a key event in the mode of action for reproductive toxicity induced by triazoles (Goetz et al., 2007). However, more mechanistic studies are needed to address the exact mechanisms behind the virilizing effects of the females and also to further address which steps in the steroid syntheses are affected and how the physiological endpoints are linked to these effects.
Small effects on hormone levels might be without clinical effects in adults because of a tightly controlled homeostasis but could have detrimental effects if they occur under vulnerable stages of reproductive development in the fetus (Sharpe, 2006; Toppari et al., 2006). Although the endocrine-disrupting potency for the individual triazole compounds seems rather low compared to the concentrations of these compounds in the environment and diet, human exposure to several azole fungicides simultaneously is likely, due to the wide use of these compounds, and since the azole fungicides share several mechanisms of action and endpoints (Birkhøj, M., Taxvig, C., Vinggaard, A.M., and Andersen, H.R.), the combined effects induced by these fungicides might be additive. Likewise, they can also add to effects induced by other environmental EDs sharing similar mechanisms (Birkhøj et al., 2004; Rajapakse et al., 2002; Silva et al., 2002). Thus, it seems important to survey and minimize the exposure of the human population to azole fungicides.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The Danish Environmental Protection Agency (7041-0335, 7041-0344) to U.H.
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ACKNOWLEDGMENTS |
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We are indebted to Birgitte Møller Plesning, Heidi Letting, Morten Andreasen, Bo Herbst, Dorte Hansen, Ulla El-Baroudy, and Lillian Sztuk for excellent technical assistance and to Maria Kristina Kiersgaard for assistance in the histopathological evaluation.
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REFERENCES |
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