Estrogenic Effects in Vitro and in Vivo of the Fungicide Fenarimol Helle Raun Andersen A,∗, Eva C

Home , Endosulfan, Fenarimol, Methiocarb

Toxicology Letters 163 (2006) 142–152

Estrogenic effects in vitro and in vivo of the fungicide fenarimol Helle Raun Andersen a,∗, Eva C. Bonefeld-Jørgensen b, Flemming Nielsen a, Kirsten Jarfeldt c, Magdalena Niepsuj Jayatissa b, Anne Marie Vinggaard c a Department of Environmental Medicine, Institute of Public Health, University of Southern Denmark, Winsløwparken 17, Dk-5000 Odense C, Denmark b Department of Environmental and Occupational Medicine, Institute of Public Health, Vennelyst Boulevard 6, Bldg. 260, Universitetsparken, University of Aarhus, Dk-8000 Aarhus C, Denmark c Danish Institute for Food and Veterinary Research, Department of Toxicology and Risk Assessment, Mørkhøj Bygade 19, Dk-2860 Søborg, Denmark Received 9 June 2005; received in revised form 7 October 2005; accepted 9 October 2005 Available online 1 December 2005

Abstract The fungicide fenarimol has the potential to induce endocrine disrupting effects via several mechanisms since it possesses both estrogenic and antiandrogenic activity and inhibits aromatase activity in cell culture studies. Hence, the integrated response of fenarimol in vivo is not easy to predict. In this study, we demonstrate that fenarimol is also estrogenic in vivo, causing signiﬁcantly increased uterine weight in ovariectomized female rats. In addition, mRNA levels of the estrogen responsive gene lactoferrin (LF) were decreased in uteri, serum FSH levels were increased, and T3 levels decreased in fenarimol-treated animals. To our knowledge, only two other pesticides (o,p-DDT and methoxychlor) have previously been reported to induce an estrogenic response in the rodent uterotrophic bioassay. A pronounced xenoestrogenicity in serum samples from rats treated with fenarimol and estradiol benzoate (E2B) separately or in combination was observed, demonstrating the usefulness of this approach for estimating the integrated internal exposure to xenoestrogens. The MCF-7 cell proliferation assay was used to investigate further the dose–response curves for the estrogenic, antiestrogenic, and aromatase inhibiting properties of fenarimol in vitro. The results indicates that fenarimol exhibits a dual effect being aromatase inhibitor at low concentrations and estrogenic at higher concentrations. © 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Fenarimol; Fungicide; Estrogen agonist; MCF-7 cell proliferation; Uterotrophic; Gene expression

1. Introduction range of CYP450 isoforms from all the inducible fami- lies including key enzymes involved in biosynthesis and Fenarimol is an organic chlorinated fungicide (Fig. 1) metabolism of steroids as for instance induction of all used widely in the production of fruit, vegetables, and steroid hydroxylations (Paolini et al., 1996) and inhibi- ornamental plants. It acts systemically by inhibiting tion of CYP19 aromatase (Hirsch et al., 1987; Sanderson ergosterol biosynthesis in fungi by blocking sterol C-14 et al., 2002; Vinggaard et al., 2000) that converts andro- demethylation. In mammalian cells, fenarimol affects a gens to estrogens. Recently, fenarimol was demonstrated to possess estrogenic and antiandrogenic activity in vitro (Andersen et al., 2002; Vinggaard et al., 1999) and to have antiandrogenic activity in rats in vivo (Vinggaard et ∗ Corresponding author. Tel.: +45 6550 3765; fax: +45 6591 1458. E-mail address: [email protected] (H.R. Andersen). al., 2005). Moreover, an antagonistic effect of fenarimol

pure), o,p-DDT (CAS no. 789-02-6; 99.8% pure) and endosulfan (CAS no. 115-29-7; 99.3% pure) were purchased from Ehrenstorfer (Augsburg, Germany).

2.2. Animal experiments

Female Wistar rats (HanTac:WH) were acquired from Taconic M&B, Ry, Denmark. Twenty-four females were ovariectomized at an age of 40 days, 14 days prior to study start. All animals were delivered 1 week prior to study start Fig. 1. Chemical structure of fenarimol [(␣-(2-chlorophenyl)-␣-(4- and upon arrival rats were housed in Bayer Makrolon type chlorophenyl)-5-pyrimidinemethanol)]. 3118 cages (Type: 80-III-420-H-MAK, Techniplast), three per cage with Tapvai bedding. They were fed Syn 8.IT (a diet on the Ah-receptor activity was observed in the human known to be free of phytoestrogens having a calorie value of hepatoma TV101L cell line (Long et al., 2003). Thus, 16.4 KJ/g) and were provided with acidified tap water ad libi- fenarimol is an example of a pesticide having multiple tum. Animal rooms were maintained on a 12-h light/dark cycle, a temperature of 22 ± 1 ◦C and a relative humidity of 55 ± 5%. mechanisms of action some of which may counteract Rats were weighed and assigned randomly to treatment groups or potentiate each other in vivo. For example, the estro- so that there were no statistically significant differences genic and antiandrogenic effects may be hypothesized to among group mean body weights. During testing rats were be counteracted by the aromatase inhibiting activity of weighed daily and visually inspected for health effects twice the compound. a day. In the present study, the estrogenic effect of fenarimol was investigated in vivo using the uterotrophic bioassay 2.3. Testing of ovariectomized female rats in rats combined with hormone analysis and markers of gene expression. Additionally, the total xenoestrogenic Four groups of ovariectomized female rats, that were 54 n activity in serum was estimated by a biomarker approach. days-old at the dosing start, were included in the study ( =6 per group). Group 1 served as negative controls and was given A dose–response curve for the estrogenic response in the peanut oil orally for 4 days. Group 2 was dosed fenarimol MCF-7 cell proliferation assay in vitro was performed (200 mg/kg/day orally) for 4 days. Group 3 was treated with and by using co-exposure with testosterone, this assay E2B (1 ␮g/kg/day s.c.) and peanut oil orally for 3 days and was also used to estimate the aromatase inhibiting effects group 4 was treated with E2B (1 ␮g/kg/day s.c.) and fenarimol of fenarimol. Testosterone is converted to 17␤-estradiol (200 mg/kg/day orally) for 3 days. The reason for the shorter (17␤-E2) by endogenous aromatase activity in MCF-7 dosage period in groups 3 and 4 was visible signs of toxicity in cells (Schmitt et al., 2001) subsequently leading to an group 4 after the combined E2B/fenarimol treatment. Hence, estrogenic response in the cells (Almstrup et al., 2002; groups 3 and 4 were not treated on day 4, but were killed after Kitawaki et al., 1993). Compounds that inhibit the con- 4 days together with the first two groups. version of testosterone to 17␤-E2 will, therefore, reduce Compounds were dissolved in peanut oil and sterile peanut the estrogenic response induced by testosterone. This oil was used for the E2B solution that was administered s.c. Oral dosing was done by gavage using a stainless steel nee- approach allows direct comparison of the fenarimol con- dle with silicone tip. The animals were held by hand during centrations inducing estrogenic and aromatase inhibiting dosing and not restrained otherwise. All compounds were effects in the MCF-7 cells. administered in a dosing volume of 2 ml/kg body weight. The E2B dose was always given a few minutes after the test com- 2. Materials and methods pound and the dosing was performed in the morning. On day 4, body weights were recorded and animals were euthanized 2.1. Test compounds using 60% CO2/40% O2 followed by exsanguination. All the animals from each group underwent a thorough autopsy. The Fenarimol (CAS no. 60168-88-9; 99.6% pure) was obtained uterus, liver and paired kidneys were dissected and weighed. from the Institute of Organic Industrial Chemistry, Warsaw, Organ weights were recorded as both absolute and relative Poland. Sterile peanut oil and 17␤-estradiol benzoate (E2B) to body weight. The uteri were placed in either 0.5 ml (ani- (CAS no. 56-53-1) in sterile peanut oil for in vivo studies mals not treated with estradiol) or 1.0 ml (estradiol-treated was obtained from the pharmacy at the Royal Veterinary and animals) RNAlater (Ambion) and stored at −80 ◦C for later Agriculture University of Denmark. 17␤-E2 (99.4% purity) gene expression analysis. Blood was collected by exsanguina- (Sigma, St. Louis, MO) and testosterone (Reference Standard tions in plain glass tubes and serum was prepared and stored at T-1268 Lot. 78F59652) (Sigma, St. Louis, MO) were used for −80 ◦C for later measurement of hormones and xenoestrogenic the in vitro studies. Methoxychlor (CAS no. 72-43-5; 98.4% activity. 144 H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152

2.4. Hormone analysis 10 s denaturation at 95 ◦C, 15 s annealing at 55 ◦C and 12 s elongation at 72 ◦C were performed. In each single LC FSH and T3 serum levels were analyzed using the tech- analysis, a calibrator positive control (cDNA pool) was nique of time-resolved fluorescense (Delfia, Wallac). rFSH run in parallel with a negative control (H2O) to correct for (IFMA, Delfia, Wallac OY, Turku, Finland) levels were ana- day-to-day variation. The PCR measurements were performed lyzed at Turku University, Finland as previously described (van in duplicate at least two or three times in separate LightCycler Casteren et al., 2000). The standard used was NIDDK standard runs using the same cDNA preparation from each animal. The FSH RP-2 obtained from the National Hormone and Pituitary data used for statistical analyses were the means of the mRNA Program, NIH, Rockville, MD. ratio above the internal control for each single animal.

2.5. Levels of ER␣,ER␤ and lactoferrin (LF) mRNA in uterus tissue 2.6. Xenoestrogenicity in rat serum

2.5.1. RNA isolation and cDNA synthesis Serum samples pooled from each treatment group were ana- The uteri were homogenized in RLT buffer (RNeasy Mini- lyzed for xenoestrogenic activity using a biomarker approach, kit, QIAGEN; 2 ml for 20–130 mg tissue, 4 ml for 130–250 mg which has been used for estimating xenoestrogenicity in human tissue) by an Ultra Turrax T25 rotor-stator homogenizer. Subse- serum samples (Rasmussen et al., 2003). Pooled serum samples quent extraction of total RNA was performed using the RNeasy from a group (N = 6) of intact non-ovariectomized rats dosed Mini-kit (QIAGEN). The quantity and quality of the purified with peanut oil for 4 days and a group (N = 6) of ovariectomized RNA was evaluated by spectrophotometry. cDNA was synthe- rats treated with E2B (1 ␮g/kg/day s.c.) for 4 days were also ␮ sised from 1.0 g of total RNA using the SuperScript Pream- included in this analysis. Xenoestrogens was extracted from plification System for First Strand cDNA Synthesis following samples of 4 ml pooled serum (or the achievable volume) using the instruction manual (Life Technologies, Roskilde, DK). solid-phase extraction (SPE); subsequently the extracted compounds were separated in a LaChrom HPLC system, equipped 2.5.2. Real-time (RT)-PCR with a Photo-Diode-Array detector, using a gradient system The mRNA levels were determined by RT-PCRusing Light- as previously described. The eluted extract was collected in Cycler (LC) technology (version 3.5) according to the protocol fractions and a selected fraction in which most known xenoe- by Roche Diagnostic. PCR reactions were performed in a total strogens (including fenarimol and E2B) elute, but which is volume of 10 ␮l including 1 ␮l cDNA and PCR master mix devoid of endogenous estrogens, was tested for estrogenic consisting of: PCR buffer (1×), magnesium chloride (5 mM), response in the MCF-7 cell proliferation assay. Before testing, ◦ dNTP (0.2 mM), primer mix (0.4 ␮M), probe mix (0.2 ␮M), the fractions were evaporated to dryness at 40 C under a gentle Taq polymerase (2.5 U) (Life Technologies, Roskilde, DK). nitrogen stream and reconstituted in 20 ␮l EtOH/DMSO/Milli- The PCR programs for ER␣,ER␤ and 18S rRNA were Q water (50:10:40 v/v/v) by vortex mixing for 30 s. Then, carried out as previously described (Hofmeister and Bonefeld- 200 ␮l charcoal-treated fetal calf serum was added and the Jorgensen, 2004; Grunfeld and Bonefeld-Jorgensen, 2004). To content was vortex mixed. Aliquots of 100 ␮l were transferred establish a standard curve the control plasmid (cleaved by Sca1 to polypropylene tubes and 900 ␮l estrogen-free medium was restriction enzyme (Biolab, Risskov DK)), carrying the cloned added. All serum samples were analyzed as undiluted samples gene fragment in question, was diluted to the concentration and in a range of dilutions to ensure that the response was within 0.1 ␮g/␮l, and used as a stock for the dilution series. The the detectable range. The solvent concentration (maximal 0.5% signals from the unknown samples were quantified using the ethanol and 0.1% DMSO) in the final samples did not affect cell standard curve, and the expression of ER␣ and ER␤ was nor- proliferation. Sterile filtering of the samples was unnecessary malized to the expression of the internal standard 18S rRNA in and the samples were prepared immediately prior to use. each run of a cDNA sample. The mRNA level of LF normal- ized to 18S rRNA was determined by relative quantification 2.7. Estrogenicity and aromatase inhibition in the MCF7 following the instructions given by the manufacturer Roche cell proliferation assay using following primer and probes: forward primer 5 ATGA- CAACTCCCACACCAA 3 (10 ␮M); reverse primer 5 Effects on MCF-7 cell proliferation was investigated as GACACATTGCTTGACCTGA 3 (10 ␮M); probe 1, 5 previously described (Andersen et al., 2002). Briefly, MCF-7 TTCAAAACTGCTGAGTCCCAAGTCCA-fluorescein 3 (BUS) cells (passage 113-120) were grown in DMEM (InVitro, (2 ␮M); Probe 2,5 LightCycler Red 640-TATGAACTCT- Denmark) containing 10% fetal calf serum (FCS) (Biological AGGGCTGCCAGAATCAT-Phospher 3 (2 ␮M). In addi- Industries Co., Beit Haemek, Israel) supplemented with 4 mM tion to primers and probes, the master mix for PCR contained glutamine, 15 mM HEPES and 54 ␮g/l gentamicin sulphate in 10% LightCycler DNA Master Hybridisation probe mix, and an atmosphere of 5% CO2/95% air under saturating humidity ◦ 3 mM and 4 mM MgCl2 for LF and 18SrRNA, respectively. at 37 C. MCF-7 cells were seeded in 96-well Nunclon Delta After an initial denaturation step at 95 ◦C for 30 s, MicroWell plates (Life Technologies, Denmark) at an initial temperature cycling was initiated. For LF; 55 cycles of concentration of 3 × 103 cells per well. After 24 h, the chemi- H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152 145

cals to be tested were added in experimental medium (phenol for the E2B plus fenarimol group were also compared to con- red-free DMEM (InVitro, Denmark) containing 10% charcoal- trols. For all data, significance was judged at p < 0.05. treated FCS (Biological Industries Co., Beit Haemek, Israel) supplemented with 4 mM glutamine, 20 mM HEPES and 0.1% 3. Results sodium bicarbonate. The test solutions were prepared from 10 mM stock solu- 3.1. In vitro effects tions in ethanol (96%; Fluka Chemie, Buchs, Switzerland) and the final solvent concentration did not exceed 0.5%. In each experiment, standard curves for 17␤-E2 (ranging from Fenarimol induced a significant estrogenic response 0.000001 to 0.01 ␮M) and for testosterone (ranging from in vitro in the MCF-7 cell proliferation assay at concen- 0.00001 to 100 ␮M) were performed. Fenarimol was tested (in trations between 3 and 25 ␮M(Fig. 2). The maximum a concentration range between 0.0001 and 25 ␮M) in the pres- response induced by 25 ␮M fenarimol corresponded to ence and absence of 0.00001 ␮M17␤-E2 (which induces half 66% of the maximum 17␤-E2 response. Greater concen- of the maximum response obtained with 0.001 ␮M17␤-E2) or trations were previously found to induce cytotoxicity in 1 ␮M testosterone (which induces approximately 65% of the MCF-7 cells (Andersen et al., 2002) and were, therefore, maximum 17␤-E2-induced response) to detect both antagonis- not included. The response induced at 25 ␮M fenari- tic and agonistic effects. The concentration range of fenarimol mol was completely blocked by 0.1 ␮M ICI 182.780 is below concentrations found to induce cytotoxicity in MCF-7 (i.e., the relative proliferative effect was reduced from cells (Andersen et al., 2002). The highest fenarimol concen- 66.1 ± 11.5 RPE to −2.2 ± 0.2 RPE), showing that the tration (25 ␮M) was also tested together with 0.1 ␮M of the pure antiestrogen ICI 182.780. For comparison of estrogenic proliferation response is ER dependent. When fenari- ␮ ␤ potency, the proliferative effect of methoxychlor, o,p-DDT, mol was tested together with 0.00001 M17 -E2 an and endosulfan (in a concentration range between 0.0001 and enhanced response was observed at fenarimol concen- 25 ␮M) were included. All analysis were performed as three trations of 6.3 and 12.5 ␮M compared to the response independent experiments performed in triplicate. induced by 17␤-E2 alone. The results are expressed as the relative proliferative effect The intrinsic aromatase activity of MCF-7 cells con- (RPE): verts testosterone to 17␤-E2 that subsequently stimulates PE − 1 cell proliferation. The response induced by testosterone RPE = test sample × 100 in which PE ␮ PE − 1 at concentrations at and above 0.01 M was significantly E2 max greater than that for untreated controls (Fig. 3). Max- = cell number (test sample) , imum response was induced at 5–10 ␮M. Above this cell number (negative control) concentration the response decreased, probably because

and PEE2max is PE of 0.001 ␮M17␤-E2 in the same experiment. of cytotoxicity. The effect of fenarimol on aromatase activity was estimated by concomitant testing of fenarimol and testosterone. A fixed concentration of 1 ␮M testosterone was chosen as this concentration induced an 2.8. Statistical analyses estrogenic response corresponding to 65% of the maximum 17␤-E2 response. Fenarimol concentrations at 0.8 EC25 and EC50 for estrogenicity in vitro were estimated by ␮ non-linear regression of the dose–response curves using the and 1.6 M significantly reduced the MCF-7 cell prolif- ␮ Sigmoidal Chapman function (3 Parameters) (SigmaPlot ver. eration induced by 1 M of testosterone (Fig. 3)asan 7.0). indication of aromatase inhibition. However, at higher Statistical analyses of the in vitro and gene expression data fenarimol concentrations (12.5 and 25 ␮M), the estro- were analysed using SPSS-11.5 software. When the overall genic effects of fenarimol became dominating leading ANOVA was significant and the corresponding F test showed to a significantly enhanced response compared to testos- variance homology, pair-wise comparisons between test and terone alone. control groups were made with Dunnett’s test (in vitro data) or the Least Significant Difference test (gene expression data). 3.2. In vivo effects The gene expression data were tested for normal distribution using descriptive statistics frequencies and the skewness test. In The fenarimol dose of 200 mg/kg/day was selected as case the normality test failed, a non-parametric Mann–Whitney Rank Sum test was performed. The in vivo data were analyzed the highest dose that we, based on available information using SAS version 8. Absolute and relative organ weights and from the literature, expected would cause no visible tox- hormone data were analyzed by Students’ t-test comparing the icity in the animals. In accordance with this, no visible fenarimol group versus controls and the E2B plus fenarimol signs of toxicity were seen in the three groups treated group versus the E2B group, respectively. Uterus weight data with peanut oil (control), fenarimol or E2B. However, 146 H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152

Fig. 2. Dose–response curves for 17␤-E2 and for fenarimol tested with and without 0.00001 ␮M17␤-E2 in the MCF-7 cell proliferation assay. Results are expressed as the relative proliferative effect (RPE) calculated as described in Section 2. Data represent the mean ± S.D. of three independent experiments performed in triplicate. (a) Indicates statistically significant difference from negative controls (p < 0.05) and (b) indicates statistically significant difference from the response induced by 0.00001 ␮M17␤-E2 alone (p < 0.05). clear signs of toxicity were observed in the female rats significant in the group receiving both fenarimol and after 3 days of treatment with E2B plus fenarimol, and E2B (Table 1). Liver weights were increased by fena- therefore it was decided to stop treatment of the E2B and rimol in ovariectomized females, whereas paired kidney the E2B plus fenarimol groups after these 3 dosages and weights were unaffected. A significant increase of uter- to kill the animals 24 h later (day 4) along with the ani- ine weight compared to control animals was observed mals from the control and the fenarimol-treated group. after treatment with fenarimol, as well as after treat- Body weights were decreased in fenarimol-treated ment with E2B alone and in combination with fenarimol ovariectomized females although the decrease was not (Table 1). Even the absolute uterine weights were statis-

Fig. 3. Dose-response curves for testosterone and for fenarimol tested with and without 1 ␮M testosterone in the MCF-7 cell proliferation assay. Results are expressed as the relative proliferative effect (RPE) calculated as described in Section 2. Data represent the mean ± S.D. of three independent experiments performed in triplicate. (a) Indicates statistically signiﬁcant difference from negative controls (p < 0.05) and (b) indicates statistically signiﬁcant difference from the response induced by 1 ␮M testosterone alone (p < 0.05). H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152 147

Table 1 Final body weights, absolute and relative organ weights, and hormone levels obtained in the uterus assay Control Fenarimol E2B E2B + fenarimol

Body weight (g) 214.3 ± 31.9 179 ± 13.4* 199.8 ± 14.9 177.3 ± 20.1 Uterus weight (g/kg) 0.41 ± 0.10 0.66 ± 0.07* 1.33 ± 0.16* 1.52 ± 0.27* Uterus weight (g) 0.087 ± 0.018 0.119 ± 0.014* 0.268 ± 0.049 0.272 ± 0.063* Liver weight (g/kg) 36.5 ± 7.6 63.6 ± 3.1* 39.0 ± 2.3 59.4 ± 3.3** Liver weight (g) 7.64 ± 1.07 11.39 ± 1.09 7.79 ± 0.79 10.53 ± 1.30 Paired kidney weight (g/kg) 6.5 ± 1.1 7.0 ± 0.4 7.1 ± 0.3 7.1 ± 0.1 Paired kidney weight (g) 1.37 ± 0.19 1.24 ± 0.06 1.41 ± 0.13 1.26 ± 0.13 FSH (ng/ml) 33.2 ± 6.7 44.5 ± 6.6* 35.3 ± 9.3 35.7 ± 9.9 T3 (nM) 3.23 ± 0.45 2.51 ± 0.27* 3.00 ± 0.36 2.69 ± 0.23

Results from ovariectomized Wistar rats treated with fenarimol alone (200 mg/kg orally) or with E2B (1 ␮g/kg s.c.) or with E2B and fenarimol 200 mg/kg together are shown. The control and fenarimol groups were dosed for 4 days, whereas the E2B and E2B + fenarimol groups were dosed for 3 days only due to toxicity. All animals were killed after 4 days. Data represent the mean ± S.D. of six animals per group. * Indicates a statistically significant difference (p < 0.05) from the control animals. ** Indicates a statistically significant difference (p < 0.05) from the E2B-treated animals. tical significantly increased in spite of the reduced body did not significantly affect serum concentrations of FSH weights caused by fenarimol. However, uterine weights and T3 24 h after last dosing. in rats from the E2B plus fenarimol group were not significantly different from the E2B-treated animals. The 3.3. Gene expression rise in uterine weight in fenarimol-treated animals was accompanied by an increase in serum FSH levels and a The relative expression of the estrogen responsive decrease in serum T3 levels. Compared to controls, treat- genes ER␣,ER␤, and LF was analyzed in the uteri tis- ment with E2B alone or in combination with fenarimol sue by real-time RT-PCR. Compared to control animals

Fig. 4. Relative quantiﬁcation by real time RT-PCR of mRNA levels of ER␣,ER␤ and LF in uterus from ovariectomized female rats treated with fenarimol (200 mg/kg, orally), E2B (1 ␮g/kg, s.c.) or E2B plus fenarimol. Controls were treated orally with the vehicle (peanut oil) for 4 days. The control and fenarimol groups were dosed for 4 days, whereas the groups administered E2B with or without fenarimol were treated for 3 days because of toxicity in the E2B plus fenarimol group. All animals were killed after 4 days. Total RNA was isolated from the uteri of 4–6 animals from each exposure group, but because of high variation in the control group, data from this and other control groups treated in exactly the same way were pooled. No ER␤ mRNA was detected in uterus from one animal in the control group and one animal from the group dosed with E2B. Because of atypically elevated LF mRNA in one animal from the fenarimol group (approximately 20 times greater than the mean) and one animal from the E2B group (approximately 200 times higher than the mean) compared to the level of other animals in the group the data from these two animals has been omitted. Data represent the mean ± S.D. of two or three independent LightCycler runs in duplicate. The numbers below the bars indicate the number of uteri used for mRNA isolation and RT-PCR analyses in the respective exposure groups. 148 H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152

Fig. 5. Xenoestrogenic activity in serum extracts from rats treated with fenarimol (200 mg/kg/day, orally) for 4 days, E2B (1 ␮g/kg/day s.c.) for 3 days or E2B plus fenarimol for 3 days. Controls were treated orally with the vehicle (peanut oil) for 4 days. For comparison, serum extracts from a group of non-ovariectomized controls and a group dosed with E2B (1 ␮g/kg/day, s.c.) for 4 days were included. Columns represent the result from one pooled serum sample from each dosage group. N = 6 for all groups. Results are expressed as RPE/ml of rat serum used for the extraction procedure. receiving peanut oil for 4 days, fenarimol caused a sig- near control level in the group dosed with E2B for 3 nificant decrease of LF mRNA, whereas the ER␣ or ER␤ days and killed the next day. The group given fenarimol mRNA levels were not significantly affected (Fig. 4). The (200 mg/kg/day orally) for 4 days had a xenoestrogenic mean mRNA level of the three genes was less in animals activity in serum corresponding to the group treated with receiving 1 ␮g/kg/day s.c. E2B (for 3 days and killed 24 h E2B for 4 days. The xenoestrogenic activity in the group later), although not significant at the 0.05 level. A trend treated with both fenarimol and E2B for 3 days (and toward depressed mRNA levels of ER␣ and LF was also killed next day) was at the same level as the group receiv- seen in animals treated with E2B in combination with ing fenarimol or E2B for 4 days (Fig. 5). fenarimol. 4. Discussion 3.4. Ex vivo biomarker of xenoestrogenic activity The estrogenic response observed in vitro for fena- One pooled serum sample from each dosage group rimol is in accordance with previous findings in the was tested for xenoestrogenic activity, i.e., activity orig- MCF-7 cell proliferation assay and ER reporter gene inating predominantly from fenarimol and/or E2B. After assays (Andersen et al., 2002;Vinggaard et al., 1999). SPE and HPLC-separation of the serum samples, a frac- The ability of fenarimol to inhibit testosterone induced tion free of endogenous estrogens was tested in the MCF- MCF-7 cell proliferation occurred at lower concen- 7 cell proliferation assay. The xenoestrogenic activity in trations (0.8–3 ␮M) than the fenarimol concentrations serum from the peanut oil treated ovariectomized con- needed to stimulate cell proliferation when tested alone trol rats was low and similar to the activity detected in (3–25 ␮M). This indicates that fenarimol acts as an aro- serum from a group of non-ovariectomized rats dosed matase inhibitor at low concentrations but has estrogenic with peanut oil illustrating that the endogenous produced properties at higher concentrations. Similar U-shaped estrogens have been efficiently removed by the proce- dose–response curves have been described for some phy- dure (Fig. 5). A group dosed with E2B (1 ␮g/kg/day toestrogens being aromatase inhibitors at low concentra- s.c.) for 4 days and killed 1–5 h later had an elevated tions but estrogenic at higher concentrations (Almstrup xenoestrogenic response while the activity was back to et al., 2002). Several pesticides identified as endocrine H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152 149 disruptors in vitro have the ability to act via several differ- ketoconazole) and specific aromatase inhibitors (e.g. ent mechanisms (Andersen et al., 2002) and this study fadrozole) were reported to delay onset of vaginal open- demonstrates that such compounds might have differ- ing (Marty et al., 1999). Hence, the integrated response ent effects at different exposure levels depending on the induced by compounds having both estrogenic and aro- dose–response curves for the individual effects. Another matase inhibiting effects is not easily predicted, since at explanation for the dual effect could be a concentration- some doses the two effects might neutralize each other dependent increase in formation of one or more estro- and at other doses one of the effects might dominate. genic metabolites of fenarimol. To our knowledge, only two other pesticides have pre- Aromatase inhibition by fenarimol has been demon- viously been reported to induce a response in the rodent strated in vitro in different assay systems. The low- uterotrophic bioassay. Doses of 100–500 mg/kg/day of est concentration reported to induce a significant effect o,p-DDT (Diel et al., 2000) or 50–100 mg/kg/day of was 0.5 ␮M in human choriocarcinoma cells (Vinggaard methoxychlor (Laws et al., 2000) administered orally et al., 2000), 2.5 ␮M in human placental micro- for 3 days to ovariectomized female rats induced a somes (Vinggaard et al., 2000), and above 30 ␮Min marked increase of the relative uterus weight compared human adrenocortical carcinoma cells (Sanderson et to controls. Like fenarimol, o,p-DDT and methoxychlor al., 2002). In the present study, a significant reduction are organic chlorinated compounds. Another organic in testosterone-induced MCF-7 cell proliferation was chlorinated pesticide, endosulfan, which also have been observed at 0.8 ␮M. Hence, the sensitivity of this assay reported to be estrogenic in vitro, did not induce an is similar to the most sensitive of the aromatase assays uterotrophic response in ovariectomized mice after 30 mentioned above. days of oral exposure to 4 mg/kg/day (Hiremath and The estrogenic response of fenarimol observed in Kaliwal, 2003). This lack of uterotrophic response vitro, was confirmed in vivo in the uterotrophic assay. was confirmed in studies in which endosulfan was Fenarimol caused a minor but statistically significant administered intraperitonally (Wade et al., 1997)or effect on uterus weights at a dose of 200 mg/kg b.w. for subcutaneously (Newbold et al., 2001) to immature 4 days. This dose caused no visible signs of toxicity rats or mice for 3 days. Comparison of the estrogenic but a reduced mean body weight compared to the con- potencies of o,p-DDT, methoxychlor, endosulfan and trol group indicates systemic toxicity. Previously, oral fenarimol in vitro evaluated in the MCF-7 proliferation administration of up to 35 mg/kg b.w. fenarimol for 3 assay (Table 2)(Andersen et al., 1999, 2002) shows days to either immature or ovariectomized female rats that o,p-DDT is the most and fenarimol the least potent was reported to provide no evidence for ER agonistic or and hence, also less potent than endosulfan indicating antagonistic effects in vivo, as indicated by no changes in no direct correlation between the in vitro potency uterine weights or levels of circulating estrogens (Hirsch and the ability to induce an uterotrophic response et al., 1987). Thus, in agreement with results from the in in vivo. Methoxychlor is metabolized to 2,2-bis (p- vitro experiments, the estrogenic effect in vivo seems hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is only evident at greater doses of fenarimol. Recently, a more potent estrogen than methoxychlor itself (Bulger fenarimol was tested in a juvenile female pubertal assay et al., 1978). A more extensive production of HPTE (George et al., 2003). Female Sprague-Dawley rats were in vivo than in MCF-7 cells in vitro might explain, orally dosed with 50 or 250 mg/kg/day from postnatal why methoxychlor is at least as potent as o,p-DDT in day 22 to 42/43. The day of vaginal opening (acceler- ated by estrogens) and the body weight adjusted uter- Table 2 ine weights were unchanged compared to controls. The Potencies of fenarimol and three other chlorinated organic pesticides discrepancy between the present study and the cited in the MCF-7 cell proliferation assay female pubertal study regarding estrogenic effects illus- LOEC (␮M) EC (␮M) EC (␮M) trates differences in sensitivity between the two model 25 50 systems to detect compounds with both estrogenic and Fenarimol 3.1 6.4 9.5 aromatase-inhibiting properties. The uterotrophic bioas- o,p-DDT 0.2 0.3 0.7 Methoxychlor 1.6 2.1 4.1 say in ovariectomized rodents is likely most sensitive to Endosulfan 1.6 1.7 2.9 the estrogenic properties of fenarimol as the major part of the physiological steroidogenesis has been removed Lowest observed effect concentration (LOEC) is the lowest concentration at which a significant (p ≤ 0.05) proliferative effect is detected from the animals. In the juvenile female pubertal onset compared to controls. EC25 and EC50 are the concentrations induc- assay, estrogenic compounds accelerate vaginal opening, ing a response corresponding to 25% and 50%, respectively, of the while compounds inhibiting steroid biosynthesis (e.g. maximum response induced by 1 nM of 17␤-E2. 150 H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152 vivo although the potency is less in vitro. It could be genetic and molecular events at the time point of tissue speculated whether the relatively higher in vivo response sampling, while the uterotrophic effect is an integrated observed for fenarimol than predicted from the in vitro growth response over 4 days. assay also could result from production of a more The xenoestrogenic activity in fractionated rat serum estrogenic metabolite in vivo. Fenarimol is extensively extracts, stripped for endogenous produced estrogens, metabolized and three major and more than 40 minor was analyzed by a biomarker approach, which has metabolites have been detected in urine and feces been used to estimate the xenoestrogenic response in from rats (WHO, 1995), but no studies on endocrine human serum (Rasmussen et al., 2003; Sonnenschein disrupting properties of fenarimol metabolites have been et al., 1995) and adipose tissue (Rivas et al., 2001). In reported. the present study, we wanted to evaluate the method Several single- and multi-generation studies of repro- by testing serum samples from animals exposed to a ductive toxicity of fenarimol have been performed known amount of xenoestrogens. Both E2B and fena- (WHO, 1995). In female rats, fenarimol induced delayed rimol caused a marked increase in the serum xenoestro- parturition, which has been suggested to be caused by an genic activity after 4 days of exposure. The low activity inhibition of aromatase resulting in maintenance of high in the group receiving three doses of E2B most likely plasma progesterone levels in pregnant rats. A reduced resulted from the nearly complete elimination of E2B fertility of male rats has been observed in several stud- from the blood following metabolic degradation during ies and it has been suggested to be due to an effect on the 24 h elapse from last dosing until the blood sam- the differentiation and expression of male sexual behav- ples were collected. This is also confirmed by the lack ior, controlled within the central nervous system (Hirsch of E2B-induced response on both serum FSH levels and et al., 1987). In the male rat, this mechanism is depen- the expression of ER and LF mRNA levels in the uterus. dent on the aromatization of testosterone to 17␤-E2 and The effects on gene expression were seen when dos- hence the main conclusion from these studies was that ing has continued until a few hours before blood/tissue the effects induced by fenarimol resulted from aromatase sampling (Bonefeld-Jorgensen EC, manuscript in prepa- inhibition. ration). However, the rats dosed with a combination of Besides the uterotrophic response, a decrease of the fenarimol and E2B for 3 days still had a high serum uterine LF mRNA level was seen in ovariectomized response 24 h later, suggesting reduced elimination or female rats after 4 days of exposure to fenarimol. In metabolism of one or both compounds. We observed addition, a tendency towards an increase of ER␤ mRNA in the HPLC-chromatograms (area under the curves at level was observed in uteri from animals treated with 280 nm, data not shown), that the concentration of fenari- fenarimol alone or fenarimol plus E2B. Previous in mol, and in particular one major metabolite, was greater vitro analyses in MCF-7 cells showed that fenarimol in serum from rats treated with the last of three doses of down-regulate the ER␣ mRNA level significantly at E2B plus fenarimol 24 h before sampling than in serum the maximum concentrations tested (50 ␮M), whereas from rats receiving the last of four doses of fenarimol ER␤ mRNA was unaffected upon exposure to fenari- few hours before sampling. Thus, the clearance rate of mol alone, but significantly increased when co-exposed fenarimol seems to be decreased considerably by con- with estradiol (Grunfeld and Bonefeld-Jorgensen, 2004). comitant E2B administration. In accordance with this, We have previously observed that 4 days of exposure the metabolism of fenarimol was previously reported to to E2B down-regulated the mRNA levels of ER␣,ER␤ occur slower at high doses than at low doses and to be and LF in rat uterus, significantly for the two latter slower in females than in males (WHO, 1995) suggest- genes (Bonefeld-Jorgensen EC, manuscript in prepara- ing an impact of estrogen on the metabolism of fenarimol tion). The lack of a significant effect of E2B on ER and and saturation or inhibition of the metabolic process at LF mRNA levels in this study can be explained by the high doses. shorter exposure period (3 days) and the lag time of When fenarimol was tested in combination with 17␤- at least 24 h between last dosage and sampling of the E2 directly in the MCF-7 proliferation assay, a reduced uteri. Increase in uterine weight depends on a tempo- response was seen at the highest fenarimol concentration ral cascade of complex molecular and cellular processes of 25 ␮M. A similar decrease at 25 ␮M was not seen in the uterus. The cascade is initiated by the transcrip- when fenarimol was tested alone (Fig. 2). It might be tion of genes into mRNA beginning 0–6 h after estrogen speculated whether the metabolism of fenarimol is also administration, while the actual uterotrophic response affected by 17␤-E2 in vitro in the MCF-7 cells leading begins 24–30 h after exposure (Owens and Ashby, 2002). to a prolonged elevation of exposure causing a cytotoxic Hence, the gene expression analysis reflects specific effect. H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152 151

In conclusion, fenarimol was estrogenic in vivo in Diel, P., Schulz, T., Smolnikar, K., Strunck, E., Vollmer, G., Michna, H., rats, inducing increased uterine weight and FSH lev- 2000. Ability of xeno- and phytoestrogens to modulate expression els and a decrease of the mRNA level of the estrogen of estrogen-sensitive genes in rat uterus: estrogenicity profiles and uterotropic activity. J. Steroid Biochem. Mol. Biol. 73, 1–10. responsive gene LF. In vitro, fenarimol acted as an aro- George, J.D., Tyl, R.W., Hamby, B.T., Myers, C.B., Marr, M.C. 2003. matase inhibitor at low concentrations but was estrogenic Assessment of Pubertal Development and Thyroid Function in at higher concentrations. Whether the change in response Juvenile Female CD (Sprague-Dawley) Rats after Exposure to is due to a dual effect of fenarimol as suggested for some Selected Chemicals Administered by Gavage on Postnatal Days phytoestrogens (Almstrup et al., 2002) or to formation of 22 to 42/43. U.S. EPA/RTI, 65U-08055.001.015.002 (http://www. epa.gov/scipoly/oscpendo/docs/edmvs/ one or more estrogenic metabolites at greater concentra- femalepubertalshortreportnov1103.pdf). tions could not be determined from this study. Likewise, Grunfeld, H.T., Bonefeld-Jorgensen, E.C., 2004. Effect of in vitro the estrogenic response observed in vivo could be caused estrogenic pesticides on human oestrogen receptor alpha and beta by fenarimol metabolites produced more extensively at mRNA levels. Toxicol. Lett. 151, 467–480. elevated doses. To further investigate this issue, in vitro Hiremath, M.B., Kaliwal, B.B., 2003. Evaluation of estrogenic activity and effect of endosulfan on biochemical constituents in ovariec- testing of the major fenarimol metabolites for estrogenic tomized (OVX) Swiss albino mice. Bull. Environ. Contam. Toxi- and aromatase inhibiting properties would be appropri- col. 71, 458–464. ate. 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