Antiandrogenic effects in short-term in vivo studies of the fungicide fenarimol

Toxicology 207 (2005) 21–34

Antiandrogenic effects in short-term in vivo studies of the fungicide fenarimol Anne Marie Vinggaarda,∗ , Helene Jacobsena , Stine Broeng Metzdorffa , Helle Raun Andersenb , Christine Nellemanna a

b

Department of Toxicology and Risk Assessment, Danish Institute for Food and Veterinary Research, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark Department of Environmental Medicine, University of Southern Denmark, Winsløwparken 17, DK-5000 Odense C, Denmark Received 22 June 2004; received in revised form 12 August 2004; accepted 12 August 2004 Available online 23 September 2004

Abstract The fungicide fenarimol has estrogenic and antiandrogenic activity and inhibits aromatase activity in vitro. We tested, whether fenarimol had antiandrogenic effects in vivo. In a Hershberger assay, fenarimol given orally to castrated testosterone-treated male rats caused markedly reduced weights of ventral prostate, seminal vesicles, musc. levator ani/bulbocavernosus, and bulbourethral glands. Qualitatively similar, but weaker, effects were also evident in intact fenarimol-exposed young adult males, except that prostates were not significantly affected. Changes in androgen-regulated gene expression were determined by real-time RTPCR in ventral prostates and fenarimol caused a pronounced decrease of prostate binding protein C3 (PBP C3), ornithin decarboxylase (ODC), and insulin-like-growth factor 1 (IGF-1) mRNA levels. The antiandogenic drug flutamide, included as a positive control, caused down-regulation of PBP C3 mRNA and up-regulation of TRPM-2 mRNA levels. Serum T4 levels were reduced after fenarimol treatment and a tendency towards increased LH levels was seen. However, no effects on testosterone levels or testosterone production ex vivo could be revealed. Taken together these results indicate that fenarimol acts as an antiandrogen in vivo having effects qualitatively comparable to those of flutamide on organ level, whereas differential effects on gene expression were observed. In an additional Hershberger test, the effects of fenarimol were compared to those of estradiol benzoate, prochloraz and the aromatase inhibitor fadrozole. The data indicate a similar mode of action of fenarimol and prochloraz in the males, whereas no indications were found that the estrogenic or aromatase inhibitory properties had important impact on the effects observed in the males. Thus, it is suggested that fenarimol mediates its antiandrogenic effects at least partly via antagonism of androgen receptors. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Antiandrogen; Fenarimol; Rat; AR reporter gene assay; Reproductive organs; Gene expression

∗

Corresponding author. Tel.: +45 7234 7549; fax: +45 7234 7001. E-mail address: [email protected] (A.M. Vinggaard).

0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.08.009

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1. Introduction Research in recent years has demonstrated that several pesticides exert their endocrine disrupting effects by interfering with the androgen receptor (AR) (Kelce et al., 1998; Gray et al., 1999b). One of the first chemicals reported to be an antiandrogen was the fungicide vinclozolin (Gray et al., 1994; Kelce et al., 1994). Since then several other pesticides have been demonstrated to possess antiandrogenic activity e.g. p,p -DDE, procymidone, linuron, methoxychlor, fenitrothion, and prochloraz in vitro (Kelce et al., 1995; Sohoni and Sumpter, 1998; Maness et al., 1998; Ostby et al., 1999; Vinggaard et al., 1999b) and in vivo (Hosokawa et al., 1993; Kelce et al., 1997; Gray et al., 1999a; Ostby et al., 1999; Monosson et al., 1999; Lambright et al., 2000; Tamura et al., 2001; Vinggaard et al., 2002). A few, DDT and methoxychlor, are abandoned in the industrialized part of the world, but all others, including the recently identified antiandrogen, prochloraz (Vinggaard et al., 2002), are still in common use worldwide. Furthermore, in vitro screening of 22 commonly used pesticides has revealed that several pesticides possess the ability to block AR (Andersen et al., 2002), and in a recent screening, 66 out of 200 pesticides were found to exert inhibitory activity against transcriptional activity induced by 5␣-dihydrotestosterone (Kojima et al., 2004). Thus, there is reason to believe that future studies will reveal many more chemicals with antiandrogenic activities in vivo. Fenarimol [(␣-(2-chlorophenyl)- ␣-(4-chlorophenyl)-5-pyrimidinemethanol)] (Fig. 1) is a fungicide that is widely used in the industrialized world within horticulture and within agriculture on a wide range of fruits, vegetables, hops, and wheat (WHO, 1995). It is rapidly adsorbed onto soil and sediments and is highly persistent, but not mobile in environmental matrices (WHO, 1995). However, fenarimol bioaccumulates to a very limited degree. It belongs to the chemical class

Fig. 1. Chemical structure of fenarimol [(␣-(2-chlorophenyl)- ␣-(4chlorophenyl)-5-pyrimidinemethanol)].

of halogenated pesticides that include chemicals such as DDT, and it is a known hepatic carcinogen in rodents (Flodstr¨om et al., 1990). Fenarimol is a potent inhibitor of ergosterol synthesis in fungi by blocking sterol C-14 demethylation (Henry and Sisler, 1984). In addition fenarimol has the ability to affect a whole range of cytochrome P450 (CYP450) isoforms from all inducible families (Paolini et al., 1996) including key enzymes involved in biosynthesis and metabolism of steroids as for instance CYP19 aromatase (Mason et al., 1987). The effect on aromatase activity has been demonstrated in rats in vivo, in rat microsomes (Hirsch et al., 1987), and in JEG-3 human choriocarcinoma cells (Vinggaard et al., 2000). In addition, fenarimol has also been shown to have estrogenic activity in vitro (Vinggaard et al., 1999a; Andersen et al., 2002) and antiandrogenic activity in vitro (Andersen et al., 2002; Kojima et al., 2004). After binding of ligand, the intracellular androgen receptor regulates transcription of specific genes either by increasing or suppressing their expression (Chang et al., 1995). It is well known that lack of androgens results in an increase of testosterone-repressed prostatic message 2 (TRPM-2) expression and a decrease in prostate specific binding protein polypeptide C3 (PBP C3) expression in the ventral prostate of castrated rats (Bossyns et al., 1986; Bettuzzi et al., 1989). As examples of androgen-responsive genes we have chosen to investigate the expression of PBP C3 (Bossyns et al., 1986), TRPM-2 (Montpetit et al., 1986) and ODC (Crozat et al., 1992; Betts et al., 1997) in the ventral prostate of the rat. The first two genes have previously been shown to be affected by antiandrogens such as vinclozolin and p,p -DDE (Kelce et al., 1997; Nellemann et al., 2001), whereas prochloraz was found to inhibit the expression of PBP C3 and ODC (Vinggaard et al., 2002). In addition, the effect of fenarimol exposure on the expression of IGF-1 mRNA levels in prostates was determined as a potential marker for estrogenic effects. The purpose of the present study was to determine if fenarimol acted as an antiandrogen in vivo. The antiandrogenic effects in vivo were examined in both intact and castrated testosterone-treated males of the same age by analyzing weights of reproductive organs, serum hormone levels, and prostate gene expression. The effects obtained with fenarimol in the Hershberger assay were compared to effects of other antiandrogens (i.e.

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flutamide and prochloraz), a specific and a non-specific aromatase inhibitor (i.e. fadrozole and prochloraz), and to the estrogen, estradiol benzoate in order to elucidate the mode of action of the compound.

2. Materials and methods 2.1. Test compounds Fenarimol (CAS no. 60168-88-9; 99.6% pure) and prochloraz (CAS no. 67747-09-5; 99.6% pure) were from the Institute of Organic Industrial Chemistry, Warsaw, Poland. Testosterone propionate (CAS no. 319491) was from UniKem, Denmark, and flutamide (CAS no. F-9397) and 17␤-estradiol benzoate (CAS no. 50-50-0; 98% pure) were from Sigma-Aldrich, St. Louis, MO. Fadrozole hydrochloride was a generous gift from Novartis. Sterile peanut oil was from the pharmacy at the Royal Veterinary and Agriculture University of Denmark. 2.2. AR reporter gene assay Effects of fenarimol on androgen receptor activity were tested in a reporter gene assay based on transiently transfected CHO cells as previously described (Vinggaard et al., 2002; K¨orner et al., 2004). Twelve concentrations within the range 0.025–50 ␮M were tested in quadruplicate and experiments were repeated 4 times. Cytotoxicity experiments were performed by transfecting cells with the constitutively active androgen receptor expression vector, pSVAR13, which lacks the ligand-binding domain of the receptor. 2.3. Animal experiments 2.3.1. Test species Male Wistar rats (HanTac:WH) were acquired from Taconic M&B, Denmark. In the first experiment intact Wistar male rats (42-days-old at the dosing start) were used. In the second and third experiment Wistar males (42-days-old at the dosing start), castrated at an age of 4 weeks 14 days prior to study start, were used. All animals were delivered 1 week prior to study start and upon arrival rats were housed in Bayer Makrolon type 3118 cages (Type: 80-III-420-H-MAK, Techniplast), three per cage with Tapvai bedding. They were

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fed IT SYN 8 (a diet known to be free of phytoestrogens with a calorie value of 16.4 kJ/g) and were provided with acidified tap water ad libitum. Animal rooms were maintained on a 12 h light/dark cycle, a temperature of 22 ± 1 ◦ C and a relative humidity of 55 ± 5%. Rats were weighed and divided by randomization into treatment groups so that there were no statistically significant differences among group body weight means. During testing, rats were weighed daily and visually inspected for health effects twice a day. 2.3.2. Experimental design Experiment 1: Two groups (n = 6/group) of intact Wistar male rats were included, one served as control and the other was dosed orally with fenarimol (200 mg/kg/day) for 7 days. Experiment 2: Four groups (n = 6/group) of castrated Wistar rats were included, one of them served as negative controls while the other three were treated with testosterone propionate (0.5 mg/kg/day s.c.) alone, testosterone propionate together with flutamide given s.c. (20 mg/kg/day) or with fenarimol given orally (200 mg/kg/day). Experiment 3: Six groups (n = 6/group) of castrated Wistar rats were all treated with testosterone propionate (0.5 mg/kg/day s.c.) alone, testosterone propionate together with either flutamide given s.c. (20 mg/kg/day), estradiol benzoate given s.c. (2.5 ␮g/kg/day), prochloraz given orally (100 mg/kg/day) or fenarimol given orally (200 mg/kg/day). For all experiments, compounds were dissolved in peanut oil and sterile oil was used for all solutions that were administered s.c. All compounds were administered in a dosing volume of 2 ml/kg bw and the dosing period was 7 days for all animals. The testosterone dose was always given a few minutes after the test compound and the last dosing was performed in the morning at the day of killing the animals. Body weights were recorded and animals were euthanized using CO2 /O2 followed by exsanguinations. All the animals from each group underwent a thorough autopsy. From the males the following organs were dissected and weighed: the testis (for intact animals), ventral prostates, combined seminal vesicles and coagulating glands including fluids, musc. levator ani/bulbocavernosus, paired bulbourethral glands, paired adrenal glands, liver, paired kidneys and the thyroid (only in experiment 3). Organ

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weights were calculated as both absolute and relative to body weights. The ventral prostates from experiment 1 and 2 were put in 0.5 ml RNAlater (Ambion) and stored at −20 ◦ C until RNA isolation. Blood was collected by exsanguinations in plain glass tubes and serum was prepared and stored at −80 ◦ C until measurement of hormones. 2.4. Hormone analysis LH, T4, estradiol, and testosterone serum levels were analyzed using the technique of time-resolved fluorescense (Delfia, Wallac). rLH (IFMA, Delfia, Wallac OY, Turku, Finland) was analyzed by Pirjo Pakarinen, Turku University, Finland as previously described (Haavisto et al., 1993). The standard used was NIDDK standard rLH RP-3 obtained from the National Hormone and Pituitary Program, NIH, Rockville, MD. Testosterone was extracted from rat serum by solidphase extraction using IST Isolute C18 SPE columns of 100 mg (Mid Glamorgan, UK). The serum samples (200 ␮l) were diluted two-fold with purified water and applied to columns preconditioned and rinsed with methanol and water, respectively. Interfering substances were eluted with 2 ml methanol:water (20:80 (v/v)) and steroids were eluted with 2 × 1.2 ml methanol. The solvent in these fractions was evaporated and samples were resuspended in 100 ␮l diluent based on human serum (PerkinElmer Life Sciences, Wallac). Testosterone in these extracts was measured using commercially available FIA kits from PerkinElmer Life Sciences, Wallac. Kits from the same supplier were used for T4 determinations. Prolactin levels were analyzed using the enzyme immunoassay (BiotrakTM ) developed by Amersham. Serum samples were diluted 20 times in assay buffer and 50 ␮l of this was analyzed as recommended by the manufacturer. Testis from control and fenarimol-treated intact rats (experiment 1) were excised, decapsulated and incubated in a shaking water bath at 34 ◦ C for 3 h in DMEM/F12 medium containing 0.1% BSA with or without hCG (1 U/ml). Left testis from each animal was the control and the right testis the one that was hCG-stimulated. Vials were centrifuged at 4000 × g for 10 min and supernatants were stored at −20 ◦ C until testosterone levels were analyzed using the abovementioned method.

2.5. RNA isolation and cDNA production Ventral prostates were weighed and homogenized in RLT buffer (RNeasy mini-kit, Qiagen) by an ultra turrax rotor-stator homogenizer. Subsequent extraction of total RNA was performed using the RNeasy minikit (Qiagen) following the manufacturer’s instructions. The quantity and quality of the purified RNA was evaluated by spectrophotometry. cDNA was produced from 0.5–1.0 ␮g of total RNA using omniscript RT kit (Qiagen) and the manufacturer’s instructions. 2.6. Real-time RT-PCR Real-time RT-PCR with online detection of the PCR reaction based on fluorescence monitoring (LightCycler, Roche) was employed. We used hybridization probes (TIB MolBiol, Berlin, Germany) to monitor the amount of specific target sequence produced. The hybridization probes used for TRPM-2, PBP C3 and ODC hybridize adjacent on the PCR product and the probe with the donor fluorphor (in this case FL530 nm labelled in the 3 -end) transfers energy to the adjacent acceptor fluorphor (LC640 nm, labelled in the 5 -end). A fluorescence signal is emitted and detected in each PCR cycle. For 18S rRNA and IGF-1, Taqman probes that contain a reporter dye at the 5 -end and a quencher dye at the 3 -end is cleaved during the PCR, and fluorescence from the reporter dye is detected. Quantitative results were obtained by the cycle threshold value where a signal rises above background level. The primers were selected spanning an intron to avoid amplification of genomic DNA and by gel electrophoresis the correct size of the PCR product was checked. For the different genes reported in this paper, each PCR-reaction was optimized with concern to the MgCl2 concentration, the amount of cDNA and the optimal concentration of primers and hybridization probes. Expression of the genes coding for PBP C3, TRPM-2, ODC, and IGF-1 was compared to the steady expression of 18S rRNA. PCR was performed with 4 mM MgCl2 for PBP C3, TRPM-2, and 18S rRNA, respectively. MgCl2 (5 mM) was employed for evaluation of the genes ODC and IGF-1. For PBP C3 determination 0.3 and 0.2 ␮M of primers (5 -TTGCTGCTATGCCAGTGGTT-3 , 5 -CCTCCATCATCACGCTAACATT-3 ) and probes (FL530: 5 -AGGCTGTGAAGCAATTCAAGCAG-

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TGT-3 , LC640: 5 -TTCTAGATCAGACCGACAAGACTCTGGAAA-3 ), respectively, were used. For TRPM-2 0.7 ␮M primers (5 -CTGACCCAGCAGTACAACGA-3 , 5 -TGACACGAGAGGGGACTTCT-3 ) and 0.3 ␮M probes (FL530: 5 -TAACCTCACACAGGGCGATGACCA-3 ; LC640: 5 -ACCTTCGGGTCTCCACAGTGACAAC-3 ) were employed. For 18S rRNA 0.3 ␮M probe (5 -FAM-CCTCCGACTTTCGTTCTTGATTATGA-TAMRA-3 ) and 0.5 ␮M primers (5 -AAGACGAACCAGAGCGAAAG-3 , 5 -GGCGGGTCATGGGAATAA-3 ). For ODC (0.2 ␮M probes (FL530: 5 -CCAGTGTAATCAACCCAGCTCTGGAC-3 X and LC640: 5 GTACTTCCCATCGGACTCTGGAGTGA-3 p) and 0.6 ␮M primers (5 -CAGATGCCCGCTGTGTCTT3 ; 5 -TGACTCATCTTCATCGTCCGAG-3 ). For IGF-1 0.2 ␮M probe (5 -FAM-CAACACTCATCCACAATGCCCGTCT-TAMRA-3 ) and 0.5 ␮M primers (5 -GACCAAGGGGCTTTTACTTC-3 , 5 The PCR GCAGCGGACACAGTACATCT-3 ). program followed the manufacturer’s instructions (LightCycler-DNA Master Hybridization Probes, Roche) except that Taq start antibody (Clontech) (0.16 ␮l per 20 ␮l reaction) was incubated with the DNA Master Hybridization Probes mix at room temperature for 5 min prior to addition of the remaining components. Gene expressions were analyzed at least three times for each animal in the same cDNA preparation for all tested genes including the housekeeping gene, 18S rRNA. The mean level of mRNA for each gene for each animal was taken relative to the mean level of 18S rRNA and the relative level for each gene expression was evaluated on group basis.

2.7. Histopathalogy and androgen receptor immunohistochemistry The right epididymis from 4 animals from the control and fenarimol group (experiment 1) was at autopsy fixed in 4% buffered formaldehyde for 24 h and embedded in paraffin. Paraffin sections (3–4 ␮m) were deparaffinized and two sections of the right epididymis from each animal were examined under light microscope after staining with haematoxylin and eosin and immunostained with an antibody against the human AR, respectively. Briefly, paraffin sections were deparaffinized, and heat-induced epitope retrieval in a mi-

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crowave oven was performed for 2 × 5 min in 0.01 M citrate buffer, pH 6.0. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Non-specific background staining was eliminated with 10% normal swine serum. The tissue sections were incubated overnight at 4 ◦ C with the primary rabbit polyclonal antibody (N20:sc-816, Santa Cruz Biotechnology, San Diego, CA, USA) followed by half an hour incubation at 21 ◦ C with EnvisionTM + System (Dako, Glostrup, Denmark) and visualized by the chromogenic substrate 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO, USA). Tissue sections were counter-stained with haematoxylin mounted with Aquamount (Gurr® , BDH Laboratory Supplies, UK) and examined by light microscopy. For validation of the immunostaining, three control slides with a rat testis and a cauda epididymis were stained simultaneously: on one slide the primary antibody was omitted, on another slide the primary antibody was replaced with normal rabbit serum, and on a third slide the expression of the AR was examined. The intensity of the AR immunostaining in the nucleus of the epididymis was compared by visual inspection of the control and the fenarimol-treated male animals. 2.8. Statistical analyses All calculations and statistical analysis were generated in SAS version 8 (SAS Institute Inc., Cary, NC, USA). For comparison between the castrated group treated with testosterone and the other groups, a oneway analysis of variance was performed (General linear models procedure: Proc Glm). Organ weights were analyzed using body weight as a covariate. When the overall ANOVA was significant, lsmeans (P < 0.05) was conducted for pair-wise comparison. Nonprocessed and ln transformed data were checked for normal distribution and homogeneity of variance. If data did not fulfill these conditions, data were subjected to Kruskal–Wallis test and if significant, followed by Wilcoxon’s test for pair-wise comparison. 3. Results Antiandrogenic effects of fenarimol were tested in vitro by a transactivation assay based on transient cotransfection of CHO cells with the human AR expression vector and an MMTV-LUC reporter plasmid. Fenarimol significantly inhibited the transcriptional

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A.M. Vinggaard et al. / Toxicology 207 (2005) 21–34

Fig. 2. Fenarimol-induced androgen receptor inhibition determined in an AR reporter gene assay in CHO cells in the presence of 0.1 nM R1881. Data represent the mean ± S.D. of n = 4 performed in quadruplicate. Data for cytotoxicity measurements are shown as circles. ∗ P < 0.05.

response induced by 0.1 nM R1881 at concentrations of 6.3 ␮M and up to 50 ␮M (Fig. 2). The IC50 was determined from the non-linear regression line to be 19 ␮M. Cytotoxicity on the transactivating process caused by fenarimol was assessed by transfecting the cells with the truncated and constitutively active human pSVAR13 vector that lacks the ligand-binding domain. Fenarimol did not affect the transcriptional activity within the concentration range investigated. In the intact fenarimol-treated males the weights of the seminal vesicles, musc. levator ani/ bulbocavernosus, and bulbourethral glands were significantly decreased, whereas the other reproductive organs (testis and ventral prostate,) were unaffected (Fig. 3 and Table 1). Fenarimol administration to castrated testosterone-treated males reduced significantly both absolute and relative weights of the following tissues: ventral prostate (43%), seminal vesicles (41%), musc. levator ani/bulbocavernosus (32%), and bulbourethral glands, also called Cowpers glands (51%) (Fig. 4). The numbers in the parentheses refer to reductions of absolute weights caused by fenarimol compared to the only testosterone treated rats. As expected flutamide caused even more pronounced weight reductions of these organs (Fig. 4). Body weights were decreased in fenarimol-treated intact males, whereas unaffected by fenarimol in testosterone-treated castrated males (Tables 1–3). Liver weights were increased by fenarimol in both intact

Fig. 3. Absolute weights of ventral prostates, vesicula seminalis, musc. levator ani/bulbocavernosus and bulbourethral glands from intact male rats treated for 7 days with fenarimol (200 mg/kg orally). Mean ± S.D. (n = 6). (a) Indicates statistically significant difference from control animals (P < 0.05).

and testosterone-treated castrated males. Paired kidney weights were unaffected by the treatment, whereas the weights of the adrenals were increased in both intact and castrated males. The serum T4 level was significantly decreased by fenarimol in intact males (Table 1) and in castrated males (Table 3). In both experiment 2 and 3 fenarimol showed a tendency towards increased LH levels, which was significant in experiment 3, but not in experiment 2 due to a large variation between animals. No effects of fenarimol on neither estradiol nor prolactin serum levels could be revealed in intact or castrated Table 1 Final body weights, absolute organ weights, and hormone levels obtained in intact Wistar rats treated with fenarimol 200 mg/kg orally

Body weight (g) Liver weight (g) Paired kidney weight (g) Adrenals weight (g) Right testis (g) Left testis (g) Testosterone (nM) Estradiol (nM) LH (ng/ml) Prolactin (ng/ml) T4 (nM)

Control

Fenarimol 200 mg/kg

188 ± 5 7.95 ± 0.52 1.37 ± 0.07 0.043 ± 0.004 1.11 ± 0.08 1.12 ± 0.08 1.6 ± 1.1 0.032 ± 0.003 0.75 ± 0.51 20.4 ± 14.9 111.2 ± 21.5

171 ± 15a 13.87 ± 1.93a 1.28 ± 0.10 0.050 ± 0.005a 1.09 ± 0.13 1.11 ± 0.13 2.8 ± 2.4 0.033 ± 0.007 0.60 ± 0.41 17.3 ± 13.9 72.3 ± 13.1a

Data represent the mean ± S.D. of 6 animals per group. a Indicates a statistically significant difference (P < 0.05) from the control.

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Fig. 4. Absolute weights of ventral prostates, vesicula seminalis, musc. levator ani/bulbocavernosus and bulbourethral glands from castrated rats treated for 7 days with testosterone propionate (0.5 mg/kg s.c.) with or without flutamide (20 mg/kg s.c.) and fenarimol (200 mg/kg orally). Mean ± S.D. (n = 6). (a) Indicates statistically significant difference from castrated testosterone-treated animals (P < 0.05).

Table 2 Final body weights, absolute organ weights, and hormone levels obtained in the Hershberger assay

Body weight (g) Liver weight (g) Paired kidney weight (g) Adrenals weight (mg) Estradiol (nM) LH (ng/ml) Prolactin (ng/ml) T4 (nM)b

Castrated animals

Testo

Testo + flutamide

Testo + fenarimol

172 ± 26 8.9 ± 1.4 1.4 ± 0.2 60.2 ± 14.3 0.012 ± 0.005 15.3 ± 5.9a 22.2 ± 10.3 93.0 ± 14.7

170 ± 10 9.2 ± 0.9 1.5 ± 0.1 46.7 ± 10.3 0.013 ± 0.002 2.7 ± 3.2 42.9 ± 32.1 88.5 ± 21.5

173 ± 13 9.4 ± 0.6 1.4 ± 0.1 64.4 ± 8.0a 0.012 ± 0.002 18.5 ± 6.7a 23.4 ± 10.2 89.6 ± 14.7

171 ± 27 15.1 ± 3.1a 1.3 ± 0.2 60.7 ± 9.7a 0.033 ± 0.036 6.7 ± 4.6 26.5 ± 25.1 70.9 ± 16.8

Results from castrated Wistar rats treated with testosterone propionate alone or together with flutamide (20 mg/kg s.c.) or fenarimol 200 mg/kg orally are shown. Data represent the mean ± SD of 6 animals per group. n.d.: not detectable. a Indicates a statistically significant difference (P < 0.05) from the testosterone-treated castrated animals. b n = 4.

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Table 3 Final body weights, absolute organ weights, and hormone levels obtained from castrated Wistar rats treated with testosterone propionate alone or together with flutamide (20 mg/kg s.c.), estradiol benzoate (2.5 ␮g/kg s.c.), prochloraz (100 mg/kg orally), fenarimol (200 mg/kg orally) or fadrozol (10 mg/kg orally)

Body weight (g) Liver weight (g) Paired kidney weight (g) Adrenals weight (mg) Thyroidea (mg) Ventral prostate weight (mg) Seminal vesicles (mg) Musc. levator ani (mg) Bulbourethral glands (mg) LH (ng/ml) T4 (nM)

Testo

Testo + flut

Testo + EB

Testo + prochloraz

Testo + fenarimol

Testo + fadrozol

208 ± 10 8.5 ± 1.0 1.5 ± 0.1 44 ± 8 11 ± 4 77 ± 8 176 ± 20 232 ± 21 11 ± 2 5.2 ± 4.7 112 ± 21

199 ± 16 8.1 ± 1.1 1.4 ± 0.1 45 ± 12 11 ± 3 14 ± 2a 39 ± 4a 98 ± 18a 2 ± 0.3a 26.1 ± 8.5a 96 ± 16

198 ± 18 7.5 ± 1.0 1.4 ± 0.2 44 ± 7 9±2 76 ± 9 174 ± 33 235 ± 18 12 ± 3 4.6 ± 3.0 126 ± 13

196 ± 11 9.4 ± 0.7a 1.4 ± 0.1 42 ± 8 10 ± 2 53 ± 9a 122 ± 17a 185 ± 15a 8 ± 3a 12.3 ± 2.3 56 ± 14a

191 ± 16 14.9 ±1.2a 1.4 ± 0.1 55 ± 12a 15 ± 3a 38 ± 6a 105 ± 11a 139 ± 15a 7 ± 0.1a 14.8 ± 13.9a 82 ± 17a

197 ± 21 7.6 ± 1.3 1.4 ± 0.2 44 ± 6 10 ± 2 62 ± 8a 157 ± 17 210 ± 13 11 ± 0.2 6.8 ± 10.2b 119 ± 13b

Data represent the mean ± S.D. of 6 animals per group. a Indicates a statistically significant difference (P < 0.05) from the testosterone-treated castrated animals. b n = 4.

males (Tables 1 and 2). Flutamide gave rise to a significant 6-fold increase of the LH level in the castrated testosterone-treated animals, but did not affect the other hormone levels. Investigation of ex vivo testosterone production was performed by incubating testis from control and fenarimol-treated males with or without hCG to stimulate steroidogenesis. Testosterone productions of 0.9 ± 0.4 ng/testis and 1.3 ± 1.4 ng/testis were found for control and fenarimol-treated rats, respectively, and when the testis were stimulated with hCG the levels rose to 1.8 ± 0.9 ng/testis and 2.0 ± 1.6 ng/testis for the two groups. These results showed that no statistically significant effect of fenarimol on basal or hCG-induced testosterone production could be detected. The relative expression of the genes PBP C3, TRPM-2, ODC, and IGF-1 was analyzed in ventral prostates by real-time RT-PCR (Fig. 5). Significant decreased levels of PBP C3 and ODC mRNA were seen in castrated testosterone-treated animals given fenarimol, whereas flutamide only affected PBP C3 mRNA. No effect of fenarimol on TRPM-2 mRNA was observed, whereas flutamide significantly increased expression of this gene as expected for an antiandrogen. Fenarimol, but not flutamide, decreased the relative expression of IGF-1 mRNA. In the intact male prostates, no fenarimol-induced changes in gene expression of PBP C3 or TRPM-2 mRNA could be revealed, but a statistically significant increase in ODC mRNA level was found (P = 0.003) (data not shown).

Elongated spermatids were observed in caput epididymis in 3 out of 4 control intact animals and in 1 out of 4 fenarimol-treated animals. No other dose-related histopathological differences were observed. In both groups, cellular debris was present throughout the epididymis. As expected for 49-days-old male rats no spermazoa were present in cauda epididymis. Immunostaining of the AR was observed in the nucleus of almost all the epithelium cells lining the duct of both caput and cauda epididymis and was also seen in the nucleus of some cells in the connective tissue surrounding the duct. There were no differences in intensity of the AR-immunostaining of control and fenarimol-treated animals in experiment 1 (data not shown). In order to elucidate the mechanism of action of fenarimol an additional Hershberger test was performed that apart from fenarimol included estradiol benzoate, fadrozole, and prochloraz (Table 3). Both fenarimol and prochloraz showed the expected responses on reproductive organ weight changes, on serum T4 and LH levels. Estradiol benzoate did not cause any significant responses in this assay, and the only effect caused by fadrozole was a decrease of ventral prostate weight.

4. Discussion A detailed investigation of the in vitro transcriptional activity of fenarimol showed that it acted as an

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Fig. 5. Expression of PBP C3, TRPM-2, ODC, and IGF-1 mRNAs taken relative to expression of the house-keeping gene 18S rRNA in ventral prostates from castrated animals treated for 7 days with testosterone propionate (0.5 mg/kg s.c.) with or without flutamide (20 mg/kg s.c.) or fenarimol (200 mg/kg orally). Mean ± S.D. (n = 6). (a) Indicates statistically significant difference from castrated testosterone-treated animals (P < 0.05).

AR antagonist having an IC50 of 19 ␮M, which is in agreement with Kojima et al. (2004), who reported an IC20 for fenarimol of 7 ␮M using a similar assay system and the previously published screening data (Andersen et al., 2002). For comparison the IC50 of vinclozolin and prochloraz in our AR assay is approximately 60- and 2.4-fold lower, being around 0.3 ␮M and 7.9 ␮M (Table 4). In Table 4 the in vitro potencies of these compounds are compared to the potencies observed in vivo in the Hershberger assay. For all compounds the in vitro and in vivo experiments have been performed under identical experimental conditions in our lab. Vinclozolin and prochloraz administered at a dose of 200 mg/kg reduced prostate weights by 79% and 51%, respectively, and seminal vesicles weights with 82% and 34%, respectively. These weight reductions can be compared to those obtained after fenarimol treatment in experiment 2 that were 43% and 41% for prostate and seminal vesicles weight, respectively. Thus, the in vivo effects of fenarimol in castrated

males are approximately half of those exhibited by vinclozolin, (compared to a 60-fold difference of in vitro IC50 values) and close to those observed for prochloraz. Overall, the in vivo organ effects in the Hershberger assay of both fenarimol and prochloraz seem to be stronger than predicted from the in vitro data. In general the in vitro tests seem to predict well the compounds that will give rise to in vivo Hershberger effects, but the in vivo potency of the compounds is not predictable from the in vitro data. One explanation might be the influence of toxicokinetic factors, e.g. the gastrointestinal uptake, the tissue distribution, the cellular uptake and the rate of hepatic metabolism that affect the response in vivo but are not evaluated in vitro. It is a possibility that the antiandrogenic effect in vivo is caused by one or more metabolites of fenarimol, formed more extensively in vivo than in vitro. In general, fenarimol is rapidly absorbed in rats with peak levels about 1 h after treatment and it is rapidly metabolized with a T1/2 of 12–17 h (WHO, 1995). Given the large number of

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Table 4 IC50 values for AR inhibition in vitro obtained in the same AR reporter gene assay compared to in vivo effects on reproductive organ weights, serum LH and gene expression levels in prostates obtained in Hershberger assays Effect vitroe

IC50 in Ventral prostate weight reduction in vivo Seminal vesicles weight reduction in vivo Serum LH increase PBP C3 mRNA TRPM-2 mRNA ODC mRNA

Vinclozolina

Procymidonea ,b

Prochloraz

Fenarimol

0.3 ␮M 79% 82% 4-fold ↓ ↑ ?

1.4 ␮M 66% 69% 9-fold ↓ ↑ ?

7.9 ␮M 51%c 34%c 4-foldc ↓ ↔ ↓

19 ␮M 43% / 51%d 41% / 40%d n.s. / 3-foldd ↓ ↔ ↓

All chemicals were dosed orally at 200 mg/kg to castrated 42-day-old Wistar rats for 7 days, except where stated. n.s.: not significant. a In vivo data from Nellemann et al. (2003a). b Oral administration of 100 mg/kg/day procymidone. c Data are from Vinggaard et al. (2002). d Values represent data from experiments 2 and 3, respectively. e IC values obtained after incubation with 0.1 nM R1881in our AR reporter gene assay. 50

metabolites detected, metabolism probably occurs at more than one site on the molecule and involves several pathways. The proposed major metabolic pathways are oxidation of the pyrimidine ring, the carbinol carbon, and the chlorophenol rings (WHO, 1995). Fenarimol acts in vitro via several mechanisms of action, i.e. interaction with the AR and the ER and by affecting enzymes involved in synthesis and degradation of steroids. IC50 values for in vitro effects of fenarimol on aromatase activity has been reported to be 4.1 ␮M in rat ovarian microsomes (Hirsch et al., 1987), 10 ␮M in human placental microsomes (Vinggaard et al., 2000), and 2 ␮M in JEG-3 human choriocarcinoma cells (Vinggaard et al., 2000). The EC50 values reported for ER agonism are 10 and 14 ␮M as determined in an MCF7 cell proliferation assay (Andersen et al., 2002) and in a yeast reporter gene assay, respectively (Vinggaard et al., 1999a). For comparison the IC50 value for AR antagonism was determined here to be 19 ␮M, indicating that the compound affects aromatase activity and the ER in vitro at lower concentrations than those needed to block the AR in vitro. The effect detected in vivo is an integrated response of all the reactions affected by the compound, whereas the in vitro responses are reflecting direct actions of the compound with the receptor/enzyme, although some metabolism may take place in the cells. In order to elucidate if aromatase inhibition and/or ER agonism added to the in vivo response observed, we performed an additional Hershberger test with estradiol benzoate, the

aromatase inhibitor, fadrozole, and prochloraz, which also has antiandrogenic effects in vitro and in vivo and is known to be an aromatase inhibitor in vitro. The results on organ weight and hormone changes showed that the effects caused by fenarimol had a high degree of similarity to the effects seen for prochloraz, whereas both estradiol benzoate and fadrozole showed almost none of these effects (Table 3). These results indicate that neither the estrogenic nor the aromatase inhibiting activities of fenarimol are important explanations for the effects observed in vivo in male rats. Testolactone is an example of an aromatase inhibitor that has been tested in intact and castrated testosteronetreated rats (Vigersky et al., 1982). This compound inhibited the weights of ventral prostate and seminal vesicle both in the intact and castrated animals, and a similar inhibition was seen when the castrated animals were administered a mixture of testosterone and estradiol. Furthermore, testolactone binds to the AR in vitro. Thus, it was concluded that testolactone acted as an antiandrogen by blocking AR and that the effect was not caused by aromatase inhibition, as the effects were also evident after estradiol or dihydrotestosterone exposure. Prochloraz is another example of an aromatase inhibitor that has been identified as an antiandrogen in vitro and in vivo (Vinggaard et al., 2002). However, it cannot be excluded that another CYP450-mediated effect may be part of the mechanism of action of fenarimol. Fenarimol has previously been shown to cause testosterone hydroxylation in mice

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(Paolini et al., 1996) and it might be possible that part of the antiandrogenic effect of fenarimol is due to lowered testosterone levels. However, serum testosterone levels from intact fenarimol-treated animals did not indicate a decreased testosterone level (Table 1), and we did not find a significant effect on testosterone productions ex vivo in fenarimol-exposed intact males. In the intact young adult rats some of the reproductive organ changes seen in the Hershberger test were also observed, as fenarimol caused a decrease of all reproductive organ weights except for the prostate, whereas prochloraz only caused a decreased weight of seminal vesicles (Vinggaard et al., 2002). Thus, this animal model system seems to be less sensitive than the Hershberger test for screening of antiandrogenic effects. However, the intact males showed a greater sensitivity towards fenarimol-induced general toxicity compared to the testosterone-treated castrated males, as the body weights of intact males were 9% reduced after 200 mg/kg fenarimol, whereas no toxic effects were seen in the castrated males. Thus, this dose of 200 mg/kg is the absolute maximum dose that should be selected for short-term in vivo rat studies. Fenarimol is able to inhibit or induce several CYP450 enzymes giving rise to increased liver weights. The level of T4 was significantly decreased in intact males and castrated males, and this may be a result of an increased metabolism of T4-induced by fenarimol secondary to the liver enzyme induction. Similar reductions of T4 levels have been reported for the antiandrogens p,p -DDE (O’Connor et al., 1999) in CD and Long-Evans rats and for prochloraz in Wistar rats (Vinggaard et al., 2002). In contrast to flutamide, fenarimol did not significantly increase serum LH levels in castrated testosterone-treated rats. However, a tendency towards an increase in LH caused by fenarimol was seen. It has previously been found for prochloraz that a dosage of 250 mg/kg caused a statistically significant increase in LH, whereas a dosage of 200 mg/kg did not (Vinggaard et al., 2002). In contrast results from experiment 3 showed that 100 mg/kg prochloraz did significantly increase LH levels (Table 3). The biological variation between animals together with the number of animals in each Hershberger assay determines whether the effect turns out to be significant or not, and for both fenarimol and prochloraz it seems necessary to include more than 6 animals per group to be sure to obtain

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significant results on LH levels. Previously, vinclozolin and procymidone have been shown to increase LH levels at doses below 200 mg/kg (Nellemann et al., 2003a), whereas p,p -DDE did not affect LH at a dose of 200 mg/kg (Kelce et al., 1997). Many genes contain an androgen-responsiveelement in the promotor region and their expression is directly influenced by the amount of androgen or antiandrogen available. We analyzed mRNA levels of three genes PBP-C3, ODC, and TRPM-2 that all contain an androgen-responsive element in their promoter region. For a description of the function and regulation of these genes with references to original literature see Nellemann et al. (2003a). In the castrated testosteronetreated males, fenarimol caused a pronounced decrease of ODC mRNA and PBP C3 mRNA, whereas no effect on TRPM-2 mRNA could be detected. This is in agreement with results obtained for prochloraz in a similar assay (Vinggaard et al., 2002). The decrease in the level of PBP C3 caused by both fenarimol and prochloraz follows the predicted regulation after treatment with an antiandrogen such as flutamide or vinclozolin (Kelce et al., 1997). Compared to flutamide some deviations were observed as the TRPM-2 mRNA level was not affected by fenarimol. The TRPM-2 level has been reported being induced by both vinclozolin and p,p -DDE (Kelce et al., 1997) after 5 days of exposure. After 7 days of exposure effects have been found for vinclozolin and procymidone (Nellemann et al., 2001; Nellemann et al., 2003a). As the optimal alteration of TRPM-2 mRNA has been reported to occur after 4 days of exposure to antiandrogen (Leger et al., 1988), the observation of a potential TRPM-2 increase may have been lost after the 7 day exposure to the weak antiandrogenic fenarimol. ODC activity is known to be regulated by estrogens in the uterus (Branham et al., 1988) but has only been used in a few studies of antiandrogenic action in the rat despite the known ARE region in the promoter (Betts et al., 1997). In Nellemann et al. (2003b), it was observed that ODC was regulated by flutamide, but that a higher dose of flutamide was needed to change the expression than needed to change the expression of PBP C3 or TRPM-2. Flutamide did not significantly change the expression of ODC in this study due to a large variation between animals. The gene IGF-1 has been shown being up-regulated by estrogens in uterus tissue (Norstedt et al., 1989)

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and down-regulated by the 5␣-reductase inhibitor, finasteride, in rat prostates (Huynh et al., 2001). The effect of fenarimol was a down-regulation of IGF-1 in prostates, which may be a result of interaction with a sex steroid hormone receptor. Overall, the results show that gene expression analysis can be a valuable supplement to organ weights and hormone analysis in Hershberger assays in order to elucidate the mechanism of action of the test compound. Several single- and multigeneration studies of reproductive toxicity have shown that in rats and (at higher doses) in mice, but not in guinea pigs and rabbits, fenarimol reduced the fertility of males and the male offspring of treated females, increased gestation length, and reduced the live-born litter size of treated females (Hirsch et al., 1987; WHO, 1995). Based on the observation that the decrease in male-mediated fertility was associated with the absence of vaginal sperm at the time of mating, the effect appeared to be the result of an absence of male sexual behavior (Hirsch et al., 1986). Gray and Ostby (1998) subsequently described a dose-dependent decrease in male mating behavior of rats when fenarimol was administered daily from weaning through adulthood. These results suggest that the adverse effect of fenarimol on the fertility of male rats is due to an effect on the differentiation and expression of male sexual behavior, controlled within the central nervous system. In the rat, this mechanism is dependent on the aromatization of testosterone to 17␤-estradiol, whereas in humans it is thought mainly to involve testosterone and dihydrotestosterone. Treatment in utero of rats with fenarimol also inhibited the conversion of testosterone to 17ß-estradiol and reduced the concentration of nuclear estrogen receptors in the hypothalamic pre-optic area amygdala of fetuses and neonates (Hirsch et al., 1987). In the light of the present results that reveal also antiandrogenic effects in vivo of fenarimol, it seems conceivable that several modes and mechanisms of action may be involved in the reproductive toxicity of fenarimol. For instance might the previously found reductions in epididymis weights and prolactin levels be explained as secondary effects to AR antagonism. A relatively high dose of fenarimol was used in these short-term in vivo studies to show antiandrogenic effects, and an evaluation of these isolated results do not give rise to any concern for human reproductive

health. Fenarimol is once in a while detected in fruit and vegetables (DFA, 2002; Chun and Kang, 2003), but usually at levels below the maximum residue limits and the acceptable daily intake (ADI), so exposure via food to this single pesticide is not in itself expected to cause any human health problems. However, humans are usually exposed to mixtures of very many endocrine disrupting chemicals that together may act additively (Nellemann et al., 2003a), and this combined exposure may be a cause of concern for male reproductive health. Besides, endocrine disrupting properties of fenarimol have not been considered in the establishment of the ADI. Whether the ADI value should be adjusted will depend on more studies establishing NOAEL values for these effects. In conclusion, fenarimol proved to have antiandrogenic activity in vitro and in rats in vivo. Our studies showed clear effects of fenarimol on reproductive organs and prostate gene expression levels in males. Concerning the effects on reproductive organs fenarimol behaved like flutamide. However, in contrast to flutamide, fenarimol did not affect TRPM-2 mRNA levels and it decreased thyroid hormone levels. The effects on reproductive organs were also evident in intact male rats, although more clear-cut results were found in castrated males. It is suggested that the adverse effects in the males induced by fenarimol are mediated at least partly via antagonism of AR. The in vivo effects of fenarimol in males were predicted from the in vitro screening test, although the in vivo potency of the compound was not predictable when compared to relations between in vitro and in vivo effects of other endocrine disruptors.

Acknowledgements This study was supported by the Danish Medical Research Council (Grant No. 9801270) and by a grant from the Danish Environmental Protection Agency. We are indebted to Birgitte Møller Plesning, Rico Wellendorph Lehmann, and Karen Roswall for excellent technical assistance. Dr. Albert Brinkmann, Department of Endocrinology and Reproduction, Erasmus University, Rotterdam, the Netherlands kindly provided the pSVAR0 and the MMTV-LUC constructs. The MMTVLUC construct was originally developed by Organon BV.

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