ELSEVIER
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Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz
Peter Laier ª, Stine Broeng Metzdorffa, Julie Borch ª, Marie Louise Hagen ª, Ulla Hass ª, Sofie Christiansen a, Marta Axelstad ª, Thuri Kledal a, Majken Dalgaard ª, Chris McKinnell b, Leon J.S. Brokken ℃, Anne Marie Vinggaard a,*
a Danish Institute for Food and Veterinary Research, Department of Toxicology and Risk Assessment, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark b MRC Human Reproductive Sciences Unit, The Queen’s Medical Research Institute, 47 Little France Crescent, Old Dalkieth Road, Edinburgh EH16 4TJ, UK ” Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland
Received 31 August 2005; revised 12 October 2005; accepted 26 October 2005 Available online 10 January 2006
Abstract
The fungicide prochloraz has got multiple mechanisms of action that may influence the demasculinizing and reproductive toxic effects of the compound. In the present study, Wistar rats were dosed perinatally with prochloraz (50 and 150 mg/kg/day) from gestational day (GD) 7 to postnatal day (PND) 16. Caesarian sections were performed on selected dams at GD 21, while others were allowed to give birth to pups that were followed until PND 16. Prochloraz caused mild dysgenesis of the male external genitalia as well as reduced anogenital distance and retention of nipples in male pups. An increased anogenital distance indicated virilization of female pups. Effects on steroidogenesis in male fetuses became evident as decreased testicular and plasma levels of testosterone and increased levels of progesterone. Ex vivo synthesis of both steroid hormones was qualitatively similarly affected by prochloraz. Immunohistochemistry of fetal testes showed increased expression of 17a-hydroxylase/17,20- lyase (P450c17) and a reduction in 17ß-hydroxysteroid dehydrogenase (type 10) expression, whereas no changes in expression of genes involved in testicular steroidogenesis were observed. Increased expression of P450c17 mRNA was observed in fetal male adrenals, and the androgen- regulated genes ornithine decarboxylase, prostatic binding protein C3 as well as insulin-like growth factor I mRNA were reduced in ventral prostates PND 16. These results indicate that reduced activity of P450c17 may be a primary cause of the disrupted fetal steroidogenesis and that an altered androgen metabolism may play a role as well. In vitro studies on human adrenocortical carcinoma cells supported the findings in vivo as reduced testosterone and increased progesterone levels were observed. Overall, these results together indicate that prochloraz acts directly on the fetal testis to inhibit steroidogenesis and that this effect is exhibited at protein, and not at genomic, level. @ 2005 Elsevier Inc. All rights reserved.
Keywords: Prochloraz; Steroidogenesis; Antiandrogen; Rat; Leydig cell
Introduction
Increased incidence of human reproductive health effects in males such as malformed sex organs, poor semen quality and testicular cancer have been reported in many industrialized countries (Toppari et al., 2002). The first indication of a link to endocrine-active chemicals has recently been published, showing that feminization of sex organs in newborn boys was correlated to prenatal phthalate exposure (Swan et al., 2005).
Furthermore, previous epidemiological studies have indicated a causal connection between human exposure to pesticides and endocrine disrupting effects such as poor sperm quality (Swan et al., 2003) and increased incidence of cryptorchidism in sons of gardeners (Weidner et al., 1998).
An increasing amount of chemicals have been demonstrated to act as antiandrogens in experimental animals including the thoroughly studied pesticides linuron, procymidone, and vinclozolin together with diethylhexylphthalate (DEHP) and dibutylphthalate (DBP) (Gray et al., 1999a, 1999b, 2001; Thompson et al., 2005). Some of these antiandrogens have been banned and substituted with alternatives, but still many pesticides currently used in the industrialized world are
* Corresponding author. Fax: +45 72347698. E-mail address: amv@dfvf.dk (A.M. Vinggaard).
suspected of having various endocrine disrupting effects as shown by in vitro screening (Andersen et al., 2002).
The pesticide prochloraz is currently used in a great part of the world within agriculture and horticulture due to its fungicidal effects. Prochloraz has been shown to act via multiple mechanisms of action in vitro as it antagonizes the androgen and the estrogen receptor, agonizes the arylhydrocarbon receptor and inhibits aromatase activity (Andersen et al., 2002; Long et al., 2003; Mason et al., 1987). Additionally, in vivo studies on male rats have demonstrated prochloraz to act as an antiandrogen in the Hershberger assay, in which the lowest tested dose of 50 mg/kg was causing reduced weights of reproductive organs, down-regulated mRNA levels of androgen-regulated genes in prostates and increased serum LH levels (Vinggaard et al., 2002).
Developmental toxic effects of prochloraz have been reported in two recent studies (Vinggaard et al., 2005; Noriega et al., 2005). Perinatal exposure to 30 mg/kg prochloraz caused delayed delivery of the pups and increased nipple retention and reduced weights of bulbourethral glands in newborn male pups (Vinggaard et al., 2005). Furthermore, feminized behavior of the adult males in this study was expressed as an increased activity level and an increased intake of sweetened water, indicating that exposure during gestation and lactation causes permanent adverse effects in adulthood. In agreement with previous results reported by Wilson et al. (2004), these effects were accompanied by a marked reduction of fetal testosterone production and increased progesterone production, and it was suggested that the feminization of the male offspring was at least partly due to this mechanism of action. In male rats exposed in utero for 5 days to 31.25, 62.5, 125, and 250 mg/kg prochloraz, developmental effects in male pups such as reduced anogenital distance, nipple retention, hypospadias, severe phallus clefting and exposure of the os penis were observed together with observations that vaginal morphology was induced in males (Noriega et al., 2005). It was suggested that higher doses of prochloraz produce a profile of effects that resemble the profile produced by lower doses of androgen receptor antagonists like flutamide and vinclozolin. Overall, prochloraz is able to feminize male rat offspring after perinatal exposure, but the detailed mechanisms of actions that are involved remain to be elucidated.
Steroidogenesis and production of testosterone during gestation are critical for function and development of the male reproductive organs. In male rat fetuses, the antiandrogenic profile of phthalates such as DBP is distinct from that of typical androgen receptor antagonists such as flutamide, but they still elicit relatively similar reproductive abnormalities such as hypospadias and absent or deformed seminal vesicles and ventral prostates (Mylchreest et al., 2000). However, DBP does not seem to block the androgen receptor to a major extent but rather interfere with the androgen synthesis resulting in diminished testosterone production. Examination by quantita- tive real-time RT-PCR of the expression of various genes involved in androgen biosynthesis and signaling has formed part of the studies on mechanisms of action lying behind the antiandrogenic effects caused by DBP (Shultz et al., 2001; Barlow et al., 2003). Down-regulation of mRNA expression of
scavenger receptor class B-1 (SRB1), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3B-hydroxysteroid dehydrogenase (3ß-HSD) and 17a-hydroxylase/17,20-lyase (P450c17) by DBP in fetal testes suggests that cholesterol uptake and transport and its further conversion are potential mechanisms for the decreased testosterone synthesis (Barlow et al., 2003; Lehmann et al., 2004; Thompson et al., 2004). Consequently, it is of great interest to investigate whether these mechanisms of action play a role in the steroidogenic effects of prochloraz as well.
The purpose of the present study was to perform a detailed investigation of the mechanisms of action responsible for the prochloraz-induced effects on fetal steroidogenesis in male rat pups. Critical genes and proteins involved directly or indirectly in steroidogenesis and/or Leydig cell function were analyzed in the testes and adrenals by quantitative real-time RT-PCR, and the same method was applied to look for changes in expression of androgen-regulated genes in prostates from male pups. These investigations were supplemented with immunohistochemistry of relevant proteins and with hormone analyses in order to elucidate the mechanisms responsible for the observed effects. Finally, hormone levels were measured in mouse Leydig cells and human adrenocortical cells in order to find a suitable in vitro model that qualitatively reflects the effects of prochloraz on fetal steroidogenesis in vivo.
Methods
Test compounds
Prochloraz 99.3% pure (CAS no. 67747-09-5; N-propyl-N-[2-(2,4,6- trichlorophenoxy)ethyl]-1H-imidazole-1-carboxamide) from Institute of Organ- ic Chemistry (Warsaw, Poland) was used for the in vivo studies, while prochloraz 99.4% pure from Riedel de Häen (Seelze, Germany) was used for in vitro studies. The test compound was dissolved in peanut oil (no. P-2144) from Sigma-Aldrich (St. Louis, USA) for in vivo studies.
Animals and exposure
Fifty-six young adult nulliparous Wistar rats (HanTac: WH, Taconic M and B, Ejby, Denmark) were supplied at day 3 of pregnancy. The animals were distributed, housed and handled as previously described (Vinggaard et al., 2005). At the day after arrival, i.e. gestational day (GD) 4, animals were weighed and assigned to 3 groups of 24, 16 and 16 rats respectively with similar weight distributions. The rats were gavaged with 0, 50 mg/kg or 150 mg/kg of prochloraz from gestational day 7 to postnatal day (PND) 16. In the 3 groups, 21, 16 and 14 rats, respectively, appeared to be pregnant.
Health status of dams and delivery
Time of birth and the health status of the dams were monitored as previously described by Vinggaard et al. (2005). Among the time-mated animals, 13, 8 and 6 dams from the control, 50 mg/kg and 150 mg/kg prochloraz group, respectively, gave birth. The pups were counted, sexed and checked for anomalies. Pups found dead were investigated macroscopically for alterations when possible. The expected day of delivery, GD 22, was designated PND 0 for the pups.
Caesarian sections GD 21
Eight dams were randomly selected from each group for Caesarian section at GD21. From the control, 50 mg/kg and 150 mg/kg prochloraz group, respectively, 6, 7 and 5 dams were pregnant. The dams were weighed and
decapitated after CO2/O2 anesthesia, uterus was taken out, and the number of live fetuses, resorbtions and implantations was registered. Location in uterus, sex, nipples and any anomalies were recorded. Blood was taken from the trunk of all fetuses immediately after decapitation in heparin-coated vials for hormone analysis, and samples were pooled from each litter for both males and females. From each litter, one testis was taken for determination of testosterone (stored in -80 ℃ freezer until analysis) and one for histopathology (fixed in Bouin’s fixative). Paired testes and adrenals were taken from litters with two or more males and stored in RNAlater (Qiagen, Crawly, UK) for gene expression analyses. When there were more than two males in a litter, one testis was fixed in formalin for histopathology and immunohistochemistry, while the other testis was analyzed for progesterone. In litters with additional males, testes were taken and ex vivo testosterone production was determined (processed immediately).
Anogenital distance and nipple retention
Anogenital distance (AGD) was measured in the offspring at birth using a stereomicroscope. The analysis of AGD was performed by dividing the AGD with the cubic root of the pup body weight.
On PND 13, the pups were 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. The pups’ body weights were measured at both time points.
Section of pups PND 16
Body weights of all male pups were recorded, and they underwent a thorough autopsy at PND 16. The following organs were excised and weighed: liver, kidneys, adrenals, testes, epididymides, seminal vesicles, ventral prostate, bulbourethral glands and levator ani/bulbocavernosus muscle. Additionally, the thyroids were excised but not weighed (n = 5 to 8 males per age group per dose group).
For each litter at PND 16, the right or left testes were alternately fixed in Bouin’s fixative, while the other was frozen for analysis of testosterone levels. From litters with two or more males, paired testes, adrenals and liver were weighed and stored in RNAlater for gene expression analyses. When litters had three or more males, one testis was fixed in formalin, and one was frozen for analysis of progesterone levels. The thyroid, adrenals, epididymides, seminal vesicles and ventral prostate were fixed in formalin, and all fixed organs were embedded in paraffin and examined by light microscopy after staining with hematoxylin and eosin. Testes taken for determination of hormone levels were stored in -80 ℃ freezer until analysis.
In a supplementary in vivo study with similar animals, exposure period and other experimental conditions, 6, 7 and 8 pregnant Wistar rats, respectively, were exposed to 0, 50 and 100 mg/kg prochloraz. The ventral prostates from PND 16 males were weighed and stored in RNAlater for gene expression analyses.
Investigation of male external genitalia
The external genitalia were inspected at PND 16 in all males from all litters. The changes were scored on a scale from 1 to 4 in order to investigate whether male external genitals were feminized. The following criteria were used: Score 1: No effect. Normal genital tubercle, the urethral opening, is found at the tip of the genital tubercle, and the preputial skin is intact. In the perineal area, thick fur extends caudally from the base of the genital tubercle half the distance to the anus. A furless area circumscribes the anus. Score 2: Mild dysgenesis of the external genitalia: a small cavity on the inferior side of the genital tubercle and a minor cleft in the preputial opening are observed, estimated 0.5-1.4 on an arbitrary scale. The furless area around anus expands towards the base of the genital tubercle, but thick fur is still present at the base of the genital tubercle. Score 3: Moderate dysgenesis of the external genitalia: the preputial cleft is larger, estimated 1.5-2.4 on an arbitrary scale. The urethral opening is situated halfway down the inferior side of the genital tubercle (hypospadia). Partly furless or thin fur in the perineal area range from the base of the genital tubercle and caudally to the furless area circumscribing the anus. Score 4: Severe dysgenesis of the external genitalia: The preputial cleft is large, estimated 2.5- 3.5 on an arbitrary scale. The urethral opening is situated more than halfway
from the tip to the base. At the base of the genital tubercle, a groove extending laterally is observed (similar to control females at PND 16). Perineal area is totally furless.
Immunohistochemistry of fetal testes
Immunohistochemical staining was performed on four to five 4 um sections from each GD 21 testis kept in Bouin’s fixative from the control group and the 150 mg/kg prochloraz group. Following microwave pretreatment for 2 × 5 min in either citrate or T-EG buffer, sections were blocked for endogenous peroxidase activity in 3% H2O2 in PBS and blocked in 1% bovine serum albumin in PBS. Sections were incubated overnight at 4 ℃ with rabbit polyclonal antibodies against StAR (PA1-560, Affinity Bioreagents, Golden, CO), P450scc (AB 1244, Chemicon, Temecula, CA), 17-hydroxysteroid dehydrogenase (17B-HSD) type 10 (Biotrend Chemicals, Köln, Germany), 3ß- HSD or p450c17 (both antibodies were kind gifts from Dr. I. Mason, Edinburgh, UK). Sections were then incubated for 30 min with anti-rabbit EnVision+ (DAKO, Glostrup, Denmark), stained in DAB+ (DAKO, Glostrup, Denmark) and counterstained in Meyer’s hematoxylin. Negative controls were fetal control testis incubated with blocking serum instead of primary antibody.
Hormone analysis
Testosterone, progesterone, LH and thyroid hormones (T3, T4 and TSH) were analyzed in rat serum or testes at GD 21 and/or PND 16. Steroid hormones were extracted on IST Isolute SPE columns from the serum as previously described (Vinggaard et al., 2002; Birkhoj et al., 2004), and these hormones together with the thyroid hormones triiodothyronine (T3) and thyroxine (T4) were measured by time-resolved fluorescence using commercially available FIA kits (PerkinElmer Life Sciences, Turku, Finland). LH was analyzed at Turku University, Finland, using an immunofluorimetric assay as described by Haavisto et al. (1993). Rat TSH was determined by an enzyme immunoassay BioTrak from Amersham Biosciences (Uppsala, Sweden). Inhibin B was measured using an Inhibin B ELISA assay (Oxford Bio-Innovation, Oxford, UK) recognizing both the BB subunit and the «-subunit of inhibin B. Steroid hormones were analyzed in testes after extraction with diethyl ether. Decapsulated testes from neonatal and adult rats were placed in vials containing 100 and 500 ul water, extracted with 0.5 and 2.5 ml diethyl ether, respectively, and the procedure was repeated. Following evaporation, vials were added 100 and 500 ul diluent 1 (zero calibrator from PerkinElmer Life Sciences, Turku, Finland), respectively, and samples were vortexed and incubated for 10 min at 45 ℃ before analysis.
Ex vivo steroid production at GD 21 was determined as previously described (Vinggaard et al., 2005) except that a 5 h incubation was performed.
Gene expression levels determined by real-time RT-PCR
Tissues stored in RNAlater (paired testes, adrenals and ventral prostate) were homogenized, and total RNA was isolated using RNeasy-mini kit and RNase-Free DNase set from Qiagen (VWR International ApS, Albertslund, Denmark). Total RNA was eluted with 40 ul RNase-free water. RNA concentration and quality were determined using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE). cDNA was synthesized from 1 µg total RNA using the Omniscript Reverse Transcription kit with T16 oligos and an 18S rRNA primer. Samples were incubated at 37 ℃ for 60 min followed by 60 ℃ for 10 min on PTC-200 Peltier Thermal Cycler (MJ Research Inc., Watertown, MA, USA), and the final cDNA product was stored at -20 ℃ before quantification on the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Applera, Stockholm, Sweden) by standard TaqMan technology. Expression levels of the following genes were quantified in the testis and adrenals: scavenger receptor class B-1 (SRB1), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc) and 17a-hydroxylase/17,20-lyase (P450c17). The androgen receptor (AR) expres- sion levels were quantified in the epididymides. In the prostates, expression levels were quantified for prostatic binding protein C3 (PBP C3), ornithine decarboxylase (ODC), testosterone-repressed prostate message 2 (TRPM-2), insulin-like growth factor I (IGF-I), complement component 3 (Compl C3) and
androgen receptor (AR). For each sample, 2 ul cDNA (2.5 ng/ul) was amplified under universal thermal cycling parameters (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems) in a total reaction volume of 20 ul. Three separate amplifications were performed for each gene, and, when intra-assay variation was above 10%, additional amplifications were performed. All genes were quantified from standard curves, and expression levels of each target gene were normalized to the expression level of the housekeeping gene 18S rRNA.
Expression of Desert hedgehog (Dhh), Patched 1 (Ptc-1), Insl-3 and SF-1 in the testis was measured separately by quantitative PCR using Quanti Tect SYBR- Green PCR Kit (Qiagen) according to the manufacturer’s instructions by using the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA) with continuous fluorescence detection and normalized to the level of the housekeeping gene ribosomal protein S26 (S26).
Sequences for each primer set are listed in Table 1.
In vitro effects in the mouse Leydig tumor cell line mLTC-1
mLTC-1 cells (ATCC-CRL-2005) established from M548OP transplantable Leydig cell tumors carried in C57BL/6 mice were grown in 24-well culture plates (Costar, Corning, NY, USA) using the method previously described by Nikula et al. (1999). The cells were plated at a density of 1 × 105 cells/well, and prochloraz dissolved in DMSO was added to the cells in duplicates at 6.25, 12.5, 25, 50 and 100 µM. The incubation medium of control cells contained the same amount of DMSO (0.1%) as exposed cells. After 48 h of pre-incubation, the cells were washed with 2 ml PBS and stimulated with 0.135 IU/ml hCG (6500 IU/ mg) kindly provided by The NIDDK’s National Hormone and Pituitary Program and Dr. A.F. Parlow. After 3 h of incubation, the stimulation medium was removed and stored at -20 ℃ until assayed for progesterone as described for the plasma samples. In parallel to the hCG-stimulated cells, other cells were added a resazurin solution to test for cytotoxicity as described (Laier et al., 2003). Medium from these wells (100 ul) was transferred to black microtiter plates (Costar, Corning, NY, USA) before fluorescence was measured.
In vitro effects in human adrenocortical carcinoma cell line H295R
H295R cells (ATCC, CRL-2128) established from the pluripotent human adrenocortical carcinoma cell line were grown in 24-well culture plates (Costar, Corning, NY, USA) containing 1 ml DMEM/F12 medium (GibcoBRL Life Technologies, Paisley, UK) supplemented with 2.0% Nu- serum (BD Sciences Denmark), 1% ITS + premix (containing 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenium 1.25 mg/ml BSA and 5.35 µg/ml linoic acid; BD Sciences, Denmark) and supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 250 ng/ml amphotericin B (Fungizone) at 37 °℃ with a humidified atmosphere of 5% CO2/air. The cells were plated at a density of 2 × 105 cells/well and allowed to settle 1 day (approximately 24 h). Culture medium was removed, and new medium containing prochloraz dissolved in DMSO was added to the cells in triplicates at 0, 0.01, 0.03, 0.1, 0.3, 1 and 3 µM. The medium of control cells contained the same amount of DMSO (0.1%) as exposed cells. After 48 h of incubation, the medium was removed and stored at -20 ℃ until assayed for progesterone and testosterone as described for the plasma samples. After exposure, the cells were incubated with a resazurin solution to test for cytotoxicity towards the H295R cells as described (Laier et al., 2003). Medium from these wells (200 ul) was transferred to black microtiter plates (Costar, Corning, NY, USA) before fluorescence was measured.
Statistical analyses
Statistical evaluation of pregnancy data and litter data. The litter was generally considered the statistical unit, and the alpha level was 0.05. The results were analyzed by analyses of variance (ANOVA) using the SYSTAT PC-version software package (Systat, 1990). In order to adjust for litter effects, litter was included in the analysis of variance as a nested factor.
Statistical evaluation of hormone data. A one-way ANOVA was employed for all groups and, if significant, followed by the post hoc test Dunnett’s test. Significance was judged at P < 0.05.
| Gene | Primer set |
|---|---|
| 18S | 5'-FAM-ACC GGC GCA AGA CGA ACC AGA G-TAMRA-3' |
| rRNA | Forward, 5'-GCC GCT AGA GGT GAA ATT CTT G-3' Reverse, 5'-GAA AAC ATT CTT GGC AAA TGC TT-3' |
| SRB1 | 5'-FAM-AAA GCA TTT CTC CTG GCT GCG CAG-TAMRA-3' Forward, 5'-TCT GGT GCC CAT CAT TTA CCA-3' Reverse, 5'-AGC CCT TTT TAC TAC CAC TCC AAA-3' |
| StAR | 5'-FAM-CTG ACT CCT CTA ACT CCT GTC TGC CTA CAT GGT-TAMRA-3' |
| Forward, 5'-CCC TTG TTT GAA AAG GTC AAG TG-3' Reverse, 5'-TGA AAC GGG AAT GCT GTA GCT-3' | |
| P450scc | 5'-FAM-CCT TTA TGA AAT GGC ACA CAA CTT GAA GGT ACA-TAMRA-3' Forward, 5'-ACG ACC TCC ATG ACT CTG CAA T-3' Reverse, 5'-CTT CAG CCC GCA GCA TCT-3' |
| P450c17 | 5'-FAM-CGT CAA CCA TGG GAA TAT GTC CAC CAG A- TAMRA-3' |
| Forward, 5'-GCC ACG GGC GAC AGA A-3' | |
| Reverse, 5'-CCA AGC CTT TGT TGG GAA AA-3' | |
| AR | 5'-FAM-TCG CGA TTC TGG TAT GCT GCT GC-3' Forward, 5'-GAC ACT TGA GAT CCC GTC CT-3' Reverse, 5'-GAG CGA GCG GAA AGT TGT AG-3' |
| PBP C3 | 5'-FAM-TCA TCT AGA ATA CTG CAG CCA GAA CCA CTG G- TAMRA-3' |
| Forward, 5'-CCA TCC CCA TTT GCT GCT AT-3' Reverse, 5'-AGT CAC AGT TGA GTT AAT TGT ACC TCT AAT AAC-3' | |
| ODC | 5'-FAM-ACT CAC TGC TGT AAC ACA CAG CCT GTG CA-3' Forward, 5'-AAT GTG TGC AAG TAT CCC TTA CAG AA-3' Reverse, 5'-CAC AGC TTT GTA TCA TCC ACA TCT C-3' |
| TRPM- | 5'-FAM-AGT TTC TGA ACC AGA GCT CAC CCT TCT ACT |
| 2 | TCT G-3' |
| Forward, 5'-CTG GTT GGT CGC CAG CTA GA-3' Reverse, 5'-ATG CGG TCC CCG TTC AT-3' | |
| IGF-1 | 5'-FAM-CAA CAC TCA TCC ACA ATG CCC GTC T-3' Forward, 5'-GAC CAA GGG GCT TTT ACT TC-3' Reverse, 5'-GCA GCG GAC ACA GTA CAT CT-3' |
| Compl | 5'-FAM-CGT AGT CCA CTC CAG GCT CAC AAG-3' |
| C3 | Forward, 5'-CAG CCT GAA TGA ACG ACT AGA CA-3' Reverse, 5'-AAA ATC ATC CGA CAG CTC TAT CG-3' |
| S26 | Forward, 5'-AAGGAGAAACAACGGTCGTG-3' Reverse, 5'-GCAGGTCTGAATCGTGGTG-3' |
| Dhh | Forward, 5'-AAGCAACTTGTGCCTCTGCT-3' Reverse, 5'-TCGTCCCAACCTTCAGTCAC-3' |
| Ptc-1 | Forward, 5'-TACGTGGAGGTGGTTCATCA-3' Reverse, 5'-AACACCAAGGGCAAGAAATG-3' |
| SF-1 | Forward, 5'-GTCTGTCTCAAGTTCCTCATCCTC-3' Reverse, 5'-CTCGTTGCCCAAATGCTTAT-3' |
| INSL-3 | Forward, 5'-ACGCAGCCTGTGGACACC-3' Reverse, 5'-CAATCCGGGGGTGTTTCATT-3' |
Statistical analysis of organ weights and morphological alterations. One to four males per litter at PND 16 were used in the analysis of terminal body weight and organ weights. Non-processed and ln-transformed data were examined for normal distribution and homogeneity of variance. In order to adjust for litter effects, litter was included in the analysis of variance as an independent, random and nested factor (proc mixed, SAS version 8, SAS Institute Inc, Cary, NC, USA). Organ weights were analyzed using body weight as a covariate. When an overall significant treatment effect was observed, two- tailed comparison was performed using least square means. In cases where normal distribution and homogeneity of variance was not obtained, data were additionally tested with the non-parametric Kruskal-Wallis test. The scoring of macroscopic lesions of external genitalia was analyzed using Fisher’s Exact Test.
Results
Pregnancy and litter data
Prochloraz showed no effects on maternal weight during or after birth (Table 2). Maternal weight gain from GD 7 to GD 21 was significantly decreased at 150 mg/kg, but the maternal weight gain from GD 7 to PND 1 was not significantly affected by prochloraz. Litter sizes, birth weight of male and female offspring and sex ratios were unaffected. No effects were observed on pregnancy length, postnatal death or postimplanta- tion-perinatal loss.
Anogenital distance and nipple retention
Prochloraz affected dose-dependently the anogenital dis- tance in both male and female offspring (Table 2, Fig. 1A). At both 50 and 150 mg/kg, the male pups had significantly reduced anogenital distance compared to controls, whereas female pups had an increased anogenital distance compared to controls. Furthermore, both doses of prochloraz resulted in an increased number of nipples in male pups at PND 13 (Fig. 1B).
Hormone levels
Male fetuses from GD21 were sectioned, and levels of testicular testosterone and progesterone together with plasma testosterone, progesterone and luteinizing hormone (LH) were analyzed (Fig. 2). Testicular levels of testosterone were significantly decreased at both dose levels of prochloraz,
| Control | Prochloraz 50 mg/kg | Prochloraz 150 mg/kg | |
|---|---|---|---|
| Number of litters | 13 | 8 | 6 |
| Maternal weight gain GD7 to GD21 (g) | 93.2 ± 9.4 | 95.4 ± 12.9 | 77.8 ± 12.6ª |
| Maternal weight after birth (g) | 236.7 ± 15.1 | 234.1 ± 13.1 | 237.8 ±12.8 |
| Maternal weight gain GD7 to after birth (g) | 24.3 ± 9.4 | 18.6 ±4.5 | 14.8 ±11.2 |
| Pregnancy length (days) | 22.5 ± 0.5 | 22.9 ± 0.6 | 22.8±0.8 |
| Birth weight, (g) - males | 6.3 ± 0.4 | 6.4±0.4 | 6.5 ± 0.5 |
| Birth weight, (g) - females | 6.0 ± 0.3 | 6.1 ±0.2 | 6.2 ± 0.5 |
| Male pups (%) | 46± 14 | 41 ±9 | 44 ± 13 |
| Litter size at birth (live pups) | 10.5 ±1.5 | 11.0± 2.4 | 8.5 ±3.5 |
| Postnatal death, mean | 0.23 ± 0.60 | 0.13 ± 0.35 | 0.17 ± 0.41 |
| Postimplantation-perinatal loss (%)b | 6.2 ± 6.8 | 14.1 ±16.3 | 23.5 ±27.1 |
| AGD in males, at birth (mm/cubic root of bw) | 11.6±0.4 | 10.9 ±0.6ª | 10.2 ±0.8ª |
| AGD in females, at birth (mm/cubic root of bw) | 6.1 ± 0.21 | 6.3 ± 0.48a | 6.7 ± 0.40ª |
| Body weight, PND 13 (g)-males | 28.2 ± 3.5 | 26.1 ±3.9 | 26.9 ±3.5 |
| Body weight, PND 13 (g)-females | 27.8 ±3.7 | 25.8 ± 3.9 | 26.2 ± 3.6 |
Data represent means ± SD.
a P < 0.05 compared to control group.
b (no. of implantations - live pups at PND 6) / no. of implantations.
whereas levels of progesterone were significantly increased. Data on plasma pools from each exposure group showed a similar trend for both hormones. Testicular testosterone production ex vivo was examined at GD 21 as well, and, in agreement with the other data, a marked dose-dependent reduction was observed after both the low and the high dose prochloraz.
There was a tendency towards slightly increased LH plasma levels after prochloraz exposure, but, as the amounts of fetal blood were limited and only one pooled sample per group was available, it was not possible to test the hypothesis by doing statistical analysis. The abovementioned hormones were analyzed in serum and testes from male pups at PND 16 as well. A tendency towards reversed effects compared to the fetal effects was observed, i.e. increased levels of testicular and plasma testosterone and reduced levels of progesterone together with decreased LH plasma levels for the prochloraz-exposed pups (Table 3). The decrease in progesterone levels in males turned out to be statistically significant at 50 mg/kg.
Thyroid hormone levels were measured in blood plasma of PND 16 males and females, and a significantly reduced T4 level in prochloraz-exposed male pups (150 mg/kg) was observed (Table 3). However, T3 and TSH levels were not affected by prochloraz exposure for either sex.
Dysgenesis of external genitalia
Mild dysgenesis of the external genitalia was observed in males exposed to 50 mg/kg (mean score 1.25) and 150 mg/kg (mean score 1.28) prochloraz. In the controls, none of the males had scores ≥2 (mean score 1.0). In the rats exposed to 50 mg/kg prochloraz, 25% of the males had scores ≥2, and 14% of the males had scores ≥2 in the group exposed to 150 mg/kg prochloraz. For both prochloraz groups, these effects were statistically significantly different from controls (Fisher’s Exact Test).
Organ weight and histopathology
Body and organ weights of PND 16 male and female rat offspring are shown in Table 4. The number of pups (n) included for each group in the experiment varied as body weights and some organs were weighed for more than one pup per litter. A significantly diminished weight of both seminal vesicles and bulbourethral glands in male pups was observed at both doses. Female reproductive organs were unaffected. No dose-related histopathological effects were observed in any of the examined organs in males from GD 21 or PND 16.
Immunohistochemistry of fetal testes
Leydig cell cytoplasm was positively stained for P450c17, 17B-HSD (type 10), StAR, P450scc and 3ß-HSD at GD 21. AR immunostaining was seen in the nuclei of Leydig cells and peritubular cells at GD 21. The intensity of P450c17 immunostaining was clearly increased in Leydig cells of prochloraz-exposed animals compared to controls (Fig. 3).
13
5
A
B
AGD / cubic root of bw
12
*
*
Areolas in male pups
*
11
4
10
*
3
9
Males
8
Females
2
*
7
1
6
5
0
₮
0
50
100
150
Control
50 mg/kg
150 mg/kg
Prochloraz (mg/kg)
Prochloraz
6
14
10
Testosterone
Progesterone
A
800
Testosterone
Progesterone
B
5
12
production
production
*
8
ng testosterone / testis
*
pg progesterone / testis
ng testosterone / testis
10
*
pg progesterone / testis
4
600
8
6
3
*
400
6
*
4
2
*
*
4
200
1
2
2
*
T
0
Control
50 mg/kg
150 mg/kg
Control
50 mg/kg
150 mg/kg
0
0
Control
50 mg/kg
150 mg/kg
Control
50 mg/kg
150 mg/kg
0
200
14
400
400
Male . testo.
Male · prog.
C
Male
Female
D
Female · testo.
Female · prog.
12
pg testosterone / ml plasma
ng progesterone / ml plasma
150
300
300
10
pg LH / ml plasma
pg LH / ml plasma
8
100
200
200
6
50
4
100
100
2
ND
0
Control
50 mg/kg
150 mg/kg
Control
50 mg/kg
150 mg/kg
0
0
Control
50 mg/kg
150 mg/kg
Control
50 mg/kg
150 mg/kg
0
| Testis | Control | 50 mg/kg | 150 mg/kg |
|---|---|---|---|
| Testosterone, ng/testis | 0.30 ± 0.17 | 0.33 ± 0.28 | 0.54 ± 0.67 |
| Progesterone, ng/testis | 57.1 ±23.2 | 47.7 ± 14.5 | 50.1 ±41.0 |
| Plasma | Sex | Control | 50 mg/kg | 150 mg/kg |
|---|---|---|---|---|
| Testosterone, pg/ml | M | 67.7 ±28.1 | 72.7 ±74.0 | 123 ± 68.9 |
| F | 56.3 ± 41.2 | 51.0± 26.9 | 32.3 ± 26.2 | |
| Progesterone, ng/ml | M | 6.54 ±1.85 | 3.77 ± 1.54* | 3.82 ± 1.51 |
| F | 5.15 ± 1.84 | 3.98 ± 3.25 | 2.59 ± 1.24 | |
| Luteinizing hormone | M | 386 ± 382 | 225 ± 178 | 237 ±207 |
| (LH), pg/ml | F | 1605 ± 923 | 1257± 1413 | 2232 ±889 |
| Inhibin, nmol/l | M | 0.88 ± 0.31 | 1.24± 0.47 | 1.03 ± 0.43 |
| F | 0.78 ±0.57 | 0.57± 0.41 | 0.45 ± 0.36 | |
| Triiodothyronine (T3), ng/ml | M | 2.09 ± 0.13 | 2.13 ± 0.43 | 1.94 ± 0.11 |
| F | 1.91 ± 0.23 | 1.91 ± 0.19 | 1.87 ±0.20 | |
| Thyroxine (T4), ng/ml | M | 127 ± 19.7 | 134 ± 27.8 | 97.3 ±1.8* |
| F | 98.6 ± 12.7 | 91.4± 5.0 | 88.1 ±11.1 | |
| Thyroid stimulating hormone (TSH), nmol/l | M | 44.4 ± 20.9 | 47.8 ± 17.7 | 52.1 ± 25.7 |
| F | 28.0 ± 23.0 | 21.4± 2.10 | 50.3 ± 13.0 |
Data represent means ± SD.
One-way ANOVA.
* Statistical significance compared to controls by Dunnett’s t test (P < 0.05).
The intensity of immunostaining for 17-HSD (type 10) was reduced in prochloraz-exposed animals compared to controls (Fig. 4). No changes were observed in the intensity or localization of immunostaining for StAR, P450scc, 3ßHSD or AR.
Gene expression levels
Expression levels of genes involved in androgen biosyn- thesis and signaling and/or Leydig cell function were determined in paired testes and adrenals in male fetuses.
Prochloraz had no effect on the expression levels of the genes SRB1, StAR, P450scc and P450c17 in testis and AR in epididymides when compared to the housekeeping gene 18S at GD 21 or at PND 16 (Table 5). There was a clear tendency towards a dose-dependent up-regulation of P450c17 in fetal adrenals (GD 21) following exposure to 50 and 150 mg/kg prochloraz (Fig. 5). In PND 16, male pups expression of androgen-regulated genes was investigated in ventral prostates (Fig. 6). It was found that ODC, PBP C3 and IGF-1 mRNAs were significantly reduced in the prostates after exposure to 50 and 100 mg/kg prochloraz. In contrast, the expression levels of TRPM-2, Compl C3 and AR mRNAs were unaffected.
In vitro effects on steroidogenesis
In order to look for a suitable in vitro model that reliably detects compounds that affect steroidogenesis, a mouse Leydig cell line (mLTC-1) and a human adrenocortical carcinoma cell line (H295R) were investigated. In control and hCG-induced mLTC-1 cells, prochloraz caused a dose-dependent decrease of progesterone production (Fig. 7). Preliminary data indicate that this effect was seen irrespective of using cholera toxin, 8- bromo-cAMP or forskolin for stimulation of steroidogenesis (data not shown), indicating that the effect manifests itself at a level at or after protein kinase A activation. All stimulants caused elevated cAMP levels and consequently protein kinase A activation (data not shown).
H295R cells incubated with prochloraz showed a markedly dose-dependent reduction of testosterone production compared to control (Fig. 8). Testosterone levels were reduced by 50% with the lowest prochloraz concentration of 0.01 µM, and the two highest concentrations (1 and 3 µM) almost shut down the testosterone production. Progesterone levels were significantly increased after incubation with 0.1, 0.3, 1 and 3 µM prochloraz.
| n | Control | n | Prochloraz 50 mg/kg | n | Prochloraz 150 mg/kg | |
|---|---|---|---|---|---|---|
| Males | ||||||
| Body weights (g) | 35 | 35.2 ±4.3 | 25 | 32.2 ± 4.3 | 17 | 32.9 ± 4.9 |
| Liver (g) | 25 | 0.89 ± 0.16 | 15 | 0.85 ± 0.17 | 12 | 0.91 ± 0.19 |
| Paired kidneys (g) | 14 | 0.347 ± 0.053 | 8 | 0.321 ± 0.059 | 6 | 0.333 ± 0.067 |
| Paired adrenals (mg) | 25 | 10.5 ±1.6 | 15 | 10.5 ±1.5 | 12 | 10.5 ±1.9 |
| Right testis (mg) | 35 | 66.0 ± 8.7 | 25 | 64.8 ±8.7 | 17 | 61.9 ±11 |
| Left testis (mg) | 35 | 66.4 ± 7.3 | 25 | 65.0 ± 8.2 | 17 | 62.6 ± 9.7 |
| Paired epididymis (mg) | 25 | 27.0 ± 5.3 | 14 | 25.1 ±4.3 | 12 | 24.8 ± 4.5 |
| Seminal vesicles (mg) | 13 | 12.0± 3.1 | 8 | 8.78 ±2.3* | 6 | 9.55 ±1.5 |
| Ventral prostate (mg) | 25 | 16.5 ±3.1 | 16 | 14.7 ±3.5 | 12 | 13.0 ± 3.9 |
| M. levator ani/bulbocavernosus (mg) | 13 | 27.0 ± 6.3 | 8 | 22.2 ± 2.4 | 6 | 23.2 ± 1.9 |
| Bulbourethral glands (mg) | 13 | 2.16± 0.63 | 8 | 1.35 ± 0.47* | 6 | 0.790 ± 0.59* |
| Females | ||||||
| Body weights (g) | 26 | 34.4 ± 4.3 | 9 | 31.5 ±4.6 | 11 | 32.6 ± 4.6 |
| Ovaries (mg) | 26 | 6.09 ± 0.89 | 9 | 5.81 ± 1.4 | 11 | 6.33 ± 1.7 |
| Uterus (mg) | 26 | 24.9 ±2.6 | 9 | 22.5 ±2.9 | 11 | 23.8 ± 3.9 |
Data represent means ± SD.
One-way ANCOVA (body weight as the covariate).
* Statistical significance compared to controls (P < 0.05).
A1
B1
A2
B2
The formation of estradiol was significantly reduced at 0.1 µM and concentrations above showing a dose-dependently correla- tion. Visual inspection and experiments to measure cytotoxicity by the resazurin method showed no cytotoxicity of prochloraz up to 3 µM.
Discussion
In agreement with previous studies, prochloraz was found to feminize the male rat offspring by significantly reducing the anogenital distance after gestational exposure to 50 and 150 mg/ kg. This feminization was further confirmed by the observation of nipple retention in the male pups at PND 13. These results are in agreement with recent results demonstrating a dose- dependent reduction of AGD in male offspring after gestational exposure to 125 and 250 mg/kg prochloraz (Noriega et al., 2005). However, this effect was found only for unadjusted AGDs, whereas significance was lost after adjustment for body
weight. In this study, the AGD was corrected by the cubic root of the body weight, and statistical significance was obtained at the low dose of 50 mg/kg. This dose may be close to NOEL as Vinggaard et al. (2005) did not observe any effects on AGD with 30 mg/kg prochloraz. The increased anogenital distance observed in female pups may indicate an androgenic potential of prochloraz in females. Initial in vitro studies of prochloraz did not indicate any androgen activity mediated via AR, but the disrupted fetal steroidogenesis causing high progesterone levels may be the causal factor of this virilization of the females (http://progesterone.com/provera.html).
Previously, perinatal exposure to 30 mg/kg prochloraz has been shown to significantly affect nipple retention (Vinggaard et al., 2005), and dose dependency has been demonstrated by Noriega et al. (2005), who found a LOEL at a dose of 125 mg/ kg. As the exposure period in the latter study was GD 14-18 compared to our GD 7-PND 17, it suggests that the underlying causal events for the nipple retention observed at PND 13 has
A
B
| Proteins involved in Leydig cell function | Real-time RT-PCR (mRNA level) | Immunohistochemistry |
|---|---|---|
| SR-B1 | ↔ | |
| StAR | ↔ | ↔ |
| CYP450scc | ↔ | ↔ |
| 3ß-HSD | ↔ | |
| 17a-hydroxylase/17, | ↔ | ↑ |
| 20-lyase | ||
| 17B-HSD (type 10) | ↓ | |
| Dessert hedgehog (Dhh) | ↔ | |
| Ptc-1 (Dhh receptor) | ↔ | |
| SF-1 | ↔ | |
| INSL-3 | ++ | |
| AR | ↔ | ↔ |
- unaffected, ¡ increased or Į decreased by perinatal prochloraz exposure.
taken place during gestation but that exposure during lactation may result in a lower LOEL of this parameter.
The decreased maternal weight gain during gestation caused by 150 mg/kg prochloraz suggests that prochloraz induced maternal toxicity or decreased weight of the uterine content, i.e. number and/or weight of fetuses or placentas or both. Neither the maternal weight gain from GD 7 to the first day after birth (PND 1) nor the number of offspring per litter was significantly affected by prochloraz, but both values seem lower at 150 mg/ kg compared to control values. Therefore, the effect on maternal weight gain during gestation most probably reflects both slight maternal toxicity and slightly decreased litter size. But, overall, no pronounced maternal or fetal toxicity was evident at the selected doses.
The antiandrogenic potential of prochloraz has previously been demonstrated as it antagonized the androgen receptor in vitro (Andersen et al., 2002) and reduced weights of reproductive organs in the Hershberger assay (Vinggaard et al., 2002). These results classify prochloraz as an antiandrogen belonging to the same class of antiandrogens as vinclozolin, procymidone and p,p’-DDE, which primarily are considered to exert dihydrotestosterone (DHT)-dependent effects or affect DHT-dependent tissues (AGD, nipple retention, ventral prostate size, hypospadias) (Gray et al., 1999a). The decreased weight of the bulbourethral glands as well as the slightly feminized external genitals observed in male pups at PND 16 further supports that prochloraz affects DHT-dependent tissue. How- ever, the significant effect on seminal vesicle demonstrates that prochloraz also affects reproductive tissue that is considered mainly testosterone-dependent, thus resembling the effects of the phthalates (Gray et al., 1999a).
In male fetuses, testosterone levels were significantly decreased in the testis, while progesterone levels were increased. Concomitantly, fetal plasma levels of testosterone and progesterone were reduced and increased, respectively. Similar results have been observed previously by Noriega et al. (2005) and Vinggaard et al. (2005) and advocate for inhibition of the androgen biosynthesis in the Leydig cells at steps subsequent to progesterone formation at the level of 17x-
hydroxylase/17,20-lyase, 3B-HSD and/or 17ß-HSD. This was supported by the markedly reduced ex vivo testosterone production in fetal testis at 150 mg/kg (10-fold). Previously, a dose of 30 mg/kg given in utero did not cause any significant effect on ex vivo testosterone formation (Vinggaard et al., 2005), but prenatal exposure to 250 mg/kg prochloraz given from GD 14-18 was shown to reduce fetal testosterone production at GD 18 (Wilson et al., 2004). Circulating levels of LH in fetal blood plasma at GD 21 tended to be increased in prochloraz-exposed male pups, indicating that the pituitary- Leydig cell axis was intact and effective, giving a negative feedback at this time point. The effects on the testicular and plasma hormone levels observed at GD 21 in the present study were reversible as these prochloraz-induced effects were absent at PND 16. In contrast a tendency towards a rebound effect on testosterone, progesterone and LH was seen.
Prochloraz has a well-documented effect on aromatase activity and the consequent metabolism of testosterone to estradiol (Andersen et al., 2002; Sanderson et al., 2002), but, since preliminary data showed no effect on testicular estradiol levels in prochloraz-exposed male fetuses, this mechanism of action probably plays no major role in the fetal testes.
We demonstrated for the first time that the levels of the thyroid hormone T4 in plasma at PND 16 were significantly decreased after exposure to prochloraz. A previous study showed that prochloraz reduced both T4 and TSH levels in castrated, testosterone-treated rats, indicating a direct effect at the CNS level (Vinggaard et al., 2002). In this study, TSH levels were not affected by prochloraz, but this discrepancy may reflect different responsiveness of castrated versus intact rats.
In general, no visible histopathological effects in the testes were found, but changes in the expression of two enzymes involved in synthesis or metabolism of steroids were detected by immunohistochemistry. In fetal testis, prochloraz induced an increased expression of the P450c17 enzyme, which is responsible for the hydroxylation of progesterone in the 17a- position. This increased expression may reflect that the enzyme activity of P450c17 is inhibited which may further explain the accumulation of progesterone and marked reduction of
7e-6
P450c17 / 18S mRNA
6e-6
5e-6
4e-6
3e-6
2e-6
1e-6
T
0
Control
50 mg/kg
150 mg/kg
ODC / 18s mRNA
PBPC3 / 18S mRNA
TRPM-2 / 18S mRNA
CompIC3 / 18S mRNA
IGF-1 / 18S mRNA
AR / 18S mRNA
0.05
25
5e-3
5e-4
1e-2
1e-2
0.04
20
4e-3
4e-4
1e-2
1e-2
*
*
8e-3
8e-3
0.03
15
*
3e-3
3e-4
*
6e-3
*
6e-3
0.02
10
2e-3
2e-4
*
4e-3
4e-3
0.01
5
1e-3
1e-4
2e-3
2e-3
0.00
0
Control
50 mg/kg
100 mg/kg
0
Control
50 mg/kg
100 mg/kg
Control
50 mg/kg
100 mg/kg
0
0
Control
Control
50 mg/kg
0
50 mg/kg
100 mg/kg
100 mg/kg
Control
50 mg/kg
100 mg/kg
testosterone observed in male fetuses. Previous in vitro and in vivo studies of imidazole drugs such as ketoconazole suggest that the predominant effect on human adrenal and testicular steroidogenesis involves inhibition of P450c17 by direct interaction with the cytochrome P-450 component of the enzymes (Ayub and Levell, 1987, 1988). This is in agreement with the fact that we find an interaction at the protein level and not at mRNA level in the fetal testis. However, in the adrenals, an increased expression of P450c17 mRNA was seen, indicating that also steroidogenesis in adrenals was affected via a mechanism that differs from the one in the testes. Interestingly, current literature suggests that rat adrenals lack the expression of P450c17 (Pelletier et al., 2001). However, mRNA levels were clearly detectable and were significantly above the level of quantification for the analysis, although a general low expression was seen.
Concomitantly, immunohistochemical expression of 17ß- HSD (type 10) in fetal testis was reduced by prochloraz.
Progesterone formation (pmol/well)
800
Control
hCG
600
*
400
*
200
0
0
6
13
25
50
100
Prochloraz (uM)
17-HSD (type 10) is a major product of both fetal and adult-type Leydig cells in rodents and seems preferentially to convert 3a-androstanediol into dihydrotestosterone and estradiol into estrone. Thus, perinatal expression of this enzyme in fetal Leydig cells has been suggested to contribute to protection of these cells from estrogens and encourage androgen formation (Ivell et al., 2003; Mindnich et al., 2004). The reduced protein expression of 17B-HSD (type 10) may indicate an increased activity of the enzyme that is induced as a secondary defense mechanism to protect the testis from the lowered androgen levels caused by prochloraz.
Although some in vivo effects of prochloraz on steroido- genesis such as the disturbance in testosterone levels and reduced weight of seminal vesicles may indicate a similar mechanism of action as the phthalates, no effects of prochloraz were observed on mRNA levels of SRB1, StAR or P450scc responsible for cholesterol trafficking and metabolism as reported for DBP (Barlow et al., 2003; Thompson et al., 2004). In male rat fetuses, the decreased testicular steroidogen- esis caused by DBP has been demonstrated to precede the repressed transcription of StAR, Scarb1, P450scc and P450c17 and seems to be unique compared to the adrenals in which genes required for steroidogenesis were unaffected (Thompson et al., 2005).
The reduced expression levels of the androgen-regulated genes ODC, PBP C3 and IGF-1 observed in ventral prostate in PND 16 males after exposure to 50 and 100 mg/kg prochloraz further support the antiandrogenic effects of prochloraz and may be secondary to either AR antagonism or inhibition of testosterone formation. These findings are in agreement with previous results where expression of ODC and PBP C3 mRNA was found being reduced in ventral prostates of castrated testosterone-treated male rats treated with 50-150 mg/kg prochloraz (Vinggaard et al., 2002). Reduced expression of the IGF-1 gene has not previously been reported for prochloraz, but the fungicide fenarimol has been reported to reduce IGF-1 expression levels in a Hershberger assay (Vinggaard et al.,
18
0.30
16
Testosterone
Progesterone
Testosterone/Progesterone formation (pmol/well)
Estradiol
0.25
14
m
12
0.20
Estradiol formation (pmol/well)
10
0.15
8
6
0.10
4
毎
Ⅲ
山
0.05
2
0
0.00
Control
0.01
0.03
0.10
0.30
1.00
3.00
Prochloraz (uM)
2005). In general, fenarimol and prochloraz have shown very similar profiles regarding their effects in the Hershberger assay (Vinggaard et al., 2005) indicating that prochloraz may also have the potential to affect IGF-1. Additionally, the absence of effect on TRPM-2 expression levels found in this study is in agreement with previous findings for prochloraz and fenarimol in the Hershberger assay (Vinggaard et al., 2005).
The effect of prochloraz on steroidogenesis was investigated in murine Leydig tumor cells (mLTC-1) and progesterone was measured as the endpoint as mLTC-1 cells synthesize testosterone in very limited amounts compared to androsten- dione and progesterone. This is suggested to be due to the lack of isoform Type 3 of 17B-hydroxysteroid dehydrogenase/17- ketoreductase, which is responsible for the reduction of androstendione to testosterone (Panesar et al., 2003). Prochloraz was found to inhibit both basal and hCG-induced progesterone production in this cell line, the opposite effect on progesterone of what was seen in male fetuses in vivo. The reason for the reduced progesterone levels found in mLTC-1 cells is unknown, but obviously prochloraz affects steroidogenesis at a step before progesterone in these cells. It may be possible that prochloraz has more targets of action in the steroidogenic pathway affecting more than one step. Alternatively, the steroidogenic pathway in this cell line may deviate in more ways from physiological Leydig cells than we are aware of. Complemen- tary investigation of steroidogenic effects of prochloraz was carried out with the human adrenocortical carcinoma cell line H295R that is an in vitro model capable of complete steroidogenesis. The results confirmed the testosterone reduc- tion and progesterone increase observed in vivo and ex vivo with prochloraz and further supported that the disruptive effect of prochloraz on steroidogenesis is primarily mediated through a direct effect on the steroidogenic cells. Thus, this in vitro model may be a suitable candidate assay for future screening of compound-induced effects on steroidogenesis. Measuring additional metabolites in the androgen biosynthesis pathway
would be advantageous with this in vitro model for better understanding of the mechanisms of actions of prochloraz.
In conclusion, we have confirmed that prochloraz in addition to inhibiting AR also affects fetal steroidogenesis in male rats, suggesting that prochloraz’s mechanisms of actions cannot be compared exclusively with classical antiandrogens such as AR antagonists or phthalates. Furthermore, an inhibitory effect on expression of androgen-regulated genes in ventral prostates and thyroid hormone levels was found in neonatal males. The result obtained in vivo using hormone, immunohistochemical and gene expression analyses together with the in vitro data indicates that the inhibitory effect on androgen biosynthesis of prochloraz is primarily due to inhibition of the 17a-hydroxylase/17,20-lyase. In addition, an effect on androgen metabolism may play a role as the protein expression of 17B-HSD (type 10) was found being up- regulated. These results are important for future assessment of pesticides and other environmental contaminants with anti- androgen potential.
Acknowledgments
This study was supported by the Danish Medical Research Council (grant no. 22-03-0198 and 2107-04-0006) and by the EU-project “Endocrine Disrupters: Exploring Novel Endpoints, Exposure, Low Dose- and Mixture-Effects in Humans, Aquatic Wildlife and Laboratory Animals” (QRLT-2001-00603). We are indebted to Birgitte Møller Plesning, Rico Wellendorph Lehmann, Heidi Letting, Bo Herbst, Dorte Hansen, Ulla El- Baroudy and Trine Gejsing for excellent technical assistance.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.taap.2005.10.013.
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