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 0378-4274/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2005.10.004 H.R. Andersen et al. / Toxicology Letters 163 (2006) 142–152 143 pure), o,p-DDT (CAS no. 789-02-6; 99.8% pure) and endo- sulfan (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.