An Antiandrogenic Herbicide that Produces ... - Semantic Scholar

56, 389 –399 (2000) Copyright © 2000 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Cellular and Molecular Mechanisms of Action of Linuron: An Antiandrogenic Herbicide that Produces Reproductive Malformations in Male Rats C. Lambright,* J. Ostby,* K. Bobseine,* V. Wilson,† A. K. Hotchkiss,† P. C. Mann,‡ and L. E. Gray, Jr.* ,1 *U.S. EPA, NHEERL, Research Triangle Park, North Carolina; †North Carolina State University/U.S. EPA Cooperative Training Program, Raleigh, North Carolina; and ‡Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina Received January 11, 2000; accepted April 24, 2000

Antiandrogenic chemicals alter sex differentiation by several different mechanisms. Some, like flutamide, procymidone, or vinclozolin compete with androgens for the androgen receptor (AR), inhibit AR-DNA binding, and alter androgen-dependent gene expression in vivo and in vitro. Finasteride and some phthalate esters demasculinize male rats by inhibiting fetal androgen synthesis. Linuron, which is a weak competitive inhibitor of AR binding (reported Ki of 100 ␮M), alters sexual differentiation in an antiandrogenic manner. However, the pattern of malformations more closely resembles that produced by the phthalate esters than by vinclozolin treatment. The present study was designed to determine if linuron acted as an AR antagonist in vitro and in vivo. In vitro, we (1) confirmed the affinity of linuron for the rat AR, and found (2) that linuron binds human AR (hAR), and (3) acts as an hAR antagonist. Linuron competed with an androgen for rat prostatic AR (EC 50 ⴝ 100 –300 ␮M) and human AR (hAR) in a COS cell-binding assay (EC 50 ⴝ 20 ␮M). Linuron inhibited dihydrotestosterone (DHT)-hAR induced gene expression in CV-1 and MDA-MB-453-KB2 cells (EC 50 ⴝ 10 ␮M) at concentrations that were not cytotoxic. In short-term in vivo studies, linuron treatment reduced testosterone- and DHT-dependent tissue weights in the Hershberger assay (oral 100 mg/kg/d for 7 days, using castrateimmature-testosterone propionate-treated male rats; an assay used for decades to screen for AR agonists and antagonists) and altered the expression of androgen-regulated ventral prostate genes (oral 100 mg/kg/d for 4 days). Histological effects of in utero exposure to linuron (100 mg/kg/d, day 14 –18) or DBP (500 mg/kg/d, day 14 to postnatal day 3) on the testes and epididymides also are shown here. Taken together, these results support the hypothesis that linuron is an AR antagonist both in vivo and in vitro, but it remains to be determined if linuron alters sexual differentiation by additional mechanisms of action. The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. 1 To whom correspondence should be addressed at MD 72, Endocrinology Branch, U.S. EPA, NHEERL, RTD, RTP, NC, 27711. Fax: (919) 541-4017. E-mail: [email protected].

Key Words: androgen binding; flutamide; linuron; reproductive malformations; androgen receptor (AR) antagonists.

The urea-based herbicide linuron is an androgen receptor (AR) ligand that competitively inhibits the binding of androgens to rat prostatic AR with a Ki of 100 ␮M (Bauer et al., 1998; Cook et al., 1993; Waller et al., 1996). Chemicals like flutamide, vinclozolin, p,p⬘ DDE, and procymidone, which bind AR with higher affinity than linuron (Kelce et al., 1994; 1995; Ostby et al., 1999; Waller et al., 1996), alter differentiation of androgen-dependent tissues in the fetal male rat by acting as AR antagonists. These pesticides, or their active metabolites, compete with androgens for AR, inhibit AR-DNA binding and alter androgen-dependent gene expression in vivo and in vitro (Kelce et al., 1994; Ostby et al., 1999). Linuron also alters differentiation of androgen-dependent tissues in male rats when administered at 100 mg/kg/d to the dam during late pregnancy (Gray et al., 1999b). While it is tempting to conclude that linuron produces these developmental effects by acting as an AR antagonist, linuron fails to produce some effects in vivo that are pathognomonic of a pure AR antagonist. Unlike vinclozolin, which induces a 2-fold increase in serum testosterone (T) and a 4-fold increase in serum LH in pubertal male rats (Monosson et al., 1999), or flutamide, which increases T and LH 3-fold over control (Cook et al., 1993), linuron treatment does not increase serum LH (down 25%) or testosterone (up 12%). In adult rats, 200 mg linuron/kg (a neurotoxic dose) only increases LH by 25% (Cook et al, 1993), while administration of linuron at 100 mg/kg/day has no effect on serum hormone levels (Rehnberg et al., 1988). In addition, we observed relatively small reductions in sex-accessory-gland weights in adult rats at dosage levels up to 100 mg/kg/d and a modest 2.5-day delay in the age at puberty (preputial separation (PPS) in rats treated with linuron at 40 mg/kg/day (Rehnberg et al., 1988; Gray et al., 1999b). Hence, the ability of linuron to act as an AR antagonist in vivo is in question, not only because the effects are relatively small but also because delayed PPS and/or reduced sex accessory gland weights in the

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intact pubertal and adult male rat does not provide definitive evidence of AR antagonism. These effects also can result from alterations of CNS/hypothalamic-pituitary-gonadal function or altered steroid catabolism (Stoker et al., 2000). The pattern of reproductive malformations seen in male rat offspring differs from that produced by the AR antagonists vinclozolin (Gray et al., 1994, 1999b), procymidone (Ostby et al., 1999) and flutamide (Imperato-McGinley et al., 1992). When the developmental effects of linuron are compared to flutamide, procymidone, or vinclozolin at dosage levels that produce equivalent incidences of hypospadias and prostate agenesis, only linuron treatment causes epididymal lesions. More than half of the linuron-treated male offspring displayed epididymal and testicular malformations (agenesis of the epididymides, some fluid filled and flaccid testes). Such lesions are rare in vinclozolin- and procymidone-treated males at dosage levels as high as 200 mg/kg/d; levels that produce hypospadias on 100% of the males. If linuron is altering differentiation of the DHT-dependent tissues by acting as a weak AR antagonist, is it affecting epididymal differentiation by an additional mechanism? In addition to acting as AR antagonists, toxicants can alter mammalian sexual differentiation in an antiandrogenic manner by several other mechanisms of action, each of which produces a distinct profile of adverse effects. Finasteride demasculinizes DHT-dependent tissues in fetal male rats by inhibiting the enzyme 5␣-reductase, which converts testosterone (T) to dihydrotestosterone (DHT) in the skin, external genitalia, prostate, and other tissues but has little affect on T-dependent tissues. DEHP-treatment lowers whole fetal T levels by inhibiting fetal T synthesis by the testis (Parks et al., 1999), which in turn, alters both DHT- and T-dependent tissues and the testis, with the T-dependent being more profoundly affected than the DHT-dependent tissues. In this regard, in utero linuron treatment produces a pattern of malformations that more closely resembles that produced by the phthalate esters DEHP or DBP (Gray et al., 1999b) than those resulting from vinclozolin or finasteride exposures. Taken together, the above data indicate that linuron has weak affinity for the rat AR but it is uncertain whether this mechanism of action is displayed in vivo or is related to the effects of linuron on male sexual differentiation. The present study was designed (1) to confirm the affinity of linuron for the rat AR (as reported by Cook et al., 1993; Waller et al., 1996), (2) to determine if linuron also binds the human AR (hAR), and if it does, (3) if linuron displays antagonist or agonist activity in vitro, and (4) if this herbicide acts as an AR antagonist in vivo. We utilized a battery of in vivo and in vitro assays to assess whether linuron alters AR function. These included (1) in vitro AR competitive-binding assays with rat and human AR, (2) assays to assess DHT-induced transcriptional activation in CV-1 cells in vitro, (3) the nature of the in vivo effects on androgen-dependent organ weights and gene expression in immature- and adult-castrated-androgen-

treated male rats (Hershberger, 1953; Kelce, 1997), and (4) comparison of the effects of linuron and DBP on testicular and epididymal histology in male offspring following in utero exposures. Unlike previous investigations of the in vivo effects of linuron which are difficult to interpret because they used intact male rats, this study utilized protocols with castrateandrogen-treated pubertal and adult animals (Hershberger, 1953; Kelce et al., 1997) specifically designed to identify AR antagonists. MATERIALS AND METHODS In Vitro Studies Rat Prostate Competitive Binding Assay Rat ventral prostatic tissue was obtained from 90-day SD rats 24 h after castration. Prostates were homogenized (Polytron) on ice in TEDG (1.5 mM disodium ethylenediaminetetraacetate, 1.0 mM phemylmethyl sulfonyl fluoride, 1.0 mM sodium molybdate, 1.0 mM dithiothreitol, 10 mM Tris, 10% glycerol) at 10 ml per g tissue. Following centrifugation (30,000 ⫻ g), supernatants were pooled. Competitive binding of linuron (Crescent Chemical, Lot #70226, purity ⫽ 99.8% was used for all in vitro assays) was determined using the methods of Kelce et al (1994) with modification, using concentrations of 0, 0.312, 1.0, 3.12, 10.0, 31.2, 100, and 312 ␮M. In 4 separate replicates, linuron was incubated overnight at 4°C in the presence of 10 nM [3H] R1881 and 10 ␮M triamcinolone acetate, which had been dissolved in ethanol and dried down in 12 ⫻ 75 glass tubes. Conducting similar incubations with 100-fold molar excess unlabeled R1881 assessed nonspecific binding. All were incubated with 300 ␮l of the pooled prostate homogenate. Following incubation, ligand-bound receptor was separated from unbound ligand using 500 ␮l of 60% HAP slurry in 50 mM Tris buffer. Samples were washed 3 times with 50 mM Tris (centrifuged at 600 ⫻ g) to assure complete removal of unbound ligand. Receptor bound ligand was recovered using 2 ml of ethanol. Counts were determined using liquid scintillation counting. COS Whole-Cell hAR Binding Assay COS cell-binding assay was used to evaluate the ability of linuron at concentrations of 0, 0.5, 1.0, 5.0, 10, 15, and 20 ␮M to compete with [ 3H] 5 nM R1881 for hAR. In 4 replicates, COS cells (monkey kidney line, ATCC) were transiently transfected with the human AR expression vector pCMVhAR as described by Wong et al. (1995). COS cells were plated at 200,000 cells/well in 12-well plates and transfected with 1 ␮g of pCMVhAR (from Dr. Elizabeth Wilson, UNC at Chapel Hill) using diethylaminoethyl dextran. Twenty-four h later, cells were exposed to 5 nM [3H] R1881 in the presence and absence of varying doses of unlabeled compounds (2 h, 37°C). Nonspecific binding was determined by adding 100-fold molar excess of unlabeled R1881. Cells were washed in phosphate-buffered saline, lysed in 200 ␮l ZAP (0.13 M ethyldimethylhexadecylammonium bromide with 3% glacial acetic acid). Radioactivity of the lysate was determined by liquid scintillation counting. CV-1 Transcriptional Activation Assay The ability of linuron concentrations of 0, 0.5, 1.0, 5, 10, 15, and 20 ␮M to inhibit DHT-induced transcriptional activation was determined using CV-1 cells (monkey kidney line, ATCC) which were transiently transfected with 1 ␮g pCMVhAR and 5 ␮g MMTV-luciferase reporter using Fugene reagent (Boehringer Mannheim). In each of 4 replicates, cells were plated at 200,000 cells/60 mm dish and transfected using 5 ␮l Fugene reagent plus 95 ␮l serum-free medium per dish using Boehringer protocol. Twenty-four and 48 h after transfection, cells were exposed to 0.1 nM DHT and to indicated concentrations of compounds in DMEM-5% dextran charcoal stripped FBS. Five to 6 h after linuron exposure, cells were washed once with phosphate-buffered

LINURON: ANDROGEN RECEPTOR ANTAGONIST? saline and harvested with 500 ␮l lysis buffer (Promega). Luciferase assay was conducted using 0.05 ml of lysed cells and the relative light units were determined using a Monolight 2010 luminometer (Analytical Luminesce). Assessment of Cytotoxicity in CV-1 cells To assess cellular cytotoxicity, CV-1 cells (200,000 per 60-mm dish) were transfected with 50 ng CMV-ABC and 5 ␮g MMTV-luciferase (as above). Cells were dosed with 0.5, 1, and 10 ␮M of linuron and 0.1 nM DHT 24 and 48 h after transfection. The CMV-ABC receptor is constitutively active and induces a relatively high level of luciferase expression in the absence of ligand. In this regard, a reduction in luciferase activity by linuron would be indicative of cytotoxicity. Stable MDA-MB-453-KB2 Cell Transcriptional-Activation Assay MDA-MB-453-KB2 cells, which contain endogenous hAR, stably transfected with the MMTV.neo.luciferase gene construct (Bobseine et al., manuscript in preparation), were used to assess the ability of linuron to inhibit DHT-induced transcriptional activation. Cells were maintained in L-15 medium—10% FBS at 37°—with no CO 2. For each replicate, cells were plated at 10,000 cells/well in luminescent 96-well plates. Dosing solutions were prepared from stock ethanol at the time of dosing, by aliquoting 1 ␮l of stock solution into 1 ml of medium. When cells were attached (5– 6 h), medium was removed and replaced with dosing medium. Final linuron concentrations were 0, 1, 5, 10, 15, or 20 ␮M with 0.1 nM DHT. Control wells contained 100 ␮l/well (1 ␮l of ethanol/ml of medium). Cells were incubated overnight at 37°C. After 24 h, medium was removed from the plate by shaking, cells were washed once with 25 ␮l of phosphate-buffered saline and harvested with 25 ␮l lysis buffer (Promega) at room temperature. Relative light units (rlu) were determined using a microtiter plate luminometer (Dynex, Chantilly, VA). In Vivo Studies Animals Sprague-Dawley (SD) rats were purchased from Charles River Breeding Laboratory, Raleigh, NC. Upon receipt they were housed individually in clear plastic cages (20 ⫻ 25 ⫻ 47 cm) with heat-treated (to eliminate resins that induce liver enzymes), laboratory-grade pine shavings (Northeastern Products, Warrensburg, NY) as bedding. Animals were maintained on Purina Rat Chow (5001) and filtered tap water ad libitum. They were kept in a room with a 14:10 light/dark photoperiod (lights off at 1100 h), a temperature of 20 –24°C, and a relative humidity of 40 –50%. In the following in vivo studies, animals were dosed with linuron at 100 mg/kg/day. This dosage level was selected because (1) it is minimally toxic or nontoxic under the conditions used here, producing small effects on body weight gain, but no weight loss or induction of neurotoxicity or death (effects which are seen at 200 mg/kg/day (Cook et al., 1993; Rehnberg et al., 1988) and (2) this dosage level causes malformations in male rat offspring when administered during sexual differentiation (Gray et al., 1995, 1999a,b). In all studies, linuron or the positive controls (vinclozolin or flutamide) were administered orally on a mg/kg body-weight basis, and were adjusted daily for body weight. In Vivo Study 1: Hershberger Assay The purpose of this study was to determine if linuron produced antiandrogenic effects on testosterone- or DHT-dependent tissues in the castrate-testosterone-treated immature male rat. This assay was adapted from Hershberger et al. (1953) and was conducted as described in the EDSTAC Final Report (found on the web at the USEPA, OPPTS home page). Prior to and during treatment, male rats were housed in groups of 2 per cage. Castrated SD animals were purchased from Charles River (Raleigh) at 21 days-of-age. At 27 days-of-age, rats were weighed to the nearest 0.1 g and weight-ranked, and a homogeneous population of male rats was selected for the study by eliminating the “outliers” (i.e., the largest and smallest rats). In all studies, male rats were assigned to

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treatment groups in a manner that provided each group with similar means and variances in weight at the onset of the study. Hence, the design was a randomized complete block (initial body weight is the blocking factor) with 5–7 28-day-old male rats in each treatment group. The treatment conditions included (1) subcutaneous (sc) testosterone propionate (TP) (50 ␮g/day) plus oral vehicle-treated control group, (2) sc TP and oral linuron-treated group (100 mg/kg/day, lot #225, 100% technical grade, generously provided by Dupont Chemical Company), (3) sc TP and oral vinclozolin-treated (lot #10560: 2/91, ⬎99% purity, Crescent Chemical Co., Hauppague, NY) positive control group (200 mg/kg/day), and (4) sc and oral, oil-treated castrate group. Xenobiotics in corn oil or the corn oil vehicle alone were administered daily (2.5 ml/kg) for 7 days by gavage (from 28 to 34 days-of-age) at 0700 –1000 h. Sc injections of TP (50 ␮g/d) or vehicle alone were administered in 0.2 ml of oil at the same time on the dorsal surface, caudal to the nape of the neck but anterior to the base of the tail. On the day after the last treatment, males were anesthetized with CO 2 and body weight was recorded, the rat was euthanized by decapitation, and body, seminal vesicle plus coagulating gland (with and without fluid), ventral prostate, levator ani plus bulbocavernosus muscles, liver, kidney, adrenal, and pituitary weights were taken. During necropsy, care was taken to remove mesenteric fat with small surgical iris scissors from these tissues such that the fluid in the sex accessory glands was retained. In Vivo Study 2: Seven Days of Dosing with Adult-Castrated T-Implanted Male Rats This study was designed to determine if 7 days of linuron treatment would reduce androgen-dependent tissues in adult males, as it did in the Hershberger assay using immature males. Ninety-five-day-old male SD rats were anesthetized with halothane and castrated (by the method of Kelce et al., 1997). All groups of rats received 2.5-cm, testosterone (T)-filled silastic implants (Kelce et al., 1997 for details), except for the negative control group (castrate-no-T [empty implant]). This size implant has been shown to restore serum testosterone to a physiological level of about 1.5 ng/ml. On the 4 days after surgery, 7 castrate-T-implanted males were treated by gavage with linuron (100 mg/ kg/day in 2.5 ml corn oil per kg) at 0730 h. Five male rats were dosed by gavage with flutamide at 100 mg/kg/d as a positive control. The other 2 groups, one castrate-no-T-implant (8 males) and the other castrate-T-implanted (7 males), were dosed by gavage with the vehicle. On the day after the last dose, the males were necropsied. Body, ventral prostate, seminal vesicle (plus coagulating glands with fluid), epididymis, liver and levator ani plus bulbocavernosus muscles were weighed. In addition, serum was collected and prepared for determination of T levels by RIA, as described by Kelce et al. (1997). In Vivo Study 3: Four Days of Dosing with Adult-Castrate-T-Implanted Male Rats for TRPM2 and C3 mRNA Analyses This study was conducted using the above methods and those described by Kelce et al., 1997 for TRPM2 and C3 gene mRNA analyses. Changes in steady-state levels of TRPM2 and C3 mRNA from the ventral prostate reflect chemically induced alterations in the rate of androgen-regulated gene transcription and/or stability of the transcripts in situ (Kelce et al, 1997). The dosing duration was shortened from 7 to 4 days in order to maximize the ability to detect alterations of TRPM2 expression, an androgen-inhibited gene, which reportedly peaks within 5 days of castration or antiandrogen treatment. Each of the 4 treatment groups were treated orally at 0730 h (n ⫽ 5–7 per group). One group of castrate-T-implanted males was treated with linuron (100 mg/kg/day in 2.5 ml corn oil per kg), a second group was administered flutamide at 100 mg/kg/day as a positive control, and the other 2 groups (one castrate-noimplant and the other castrate-T-implanted) were dosed with the vehicle. On the afternoon after the fourth dose, the males were necropsied, as above. In addition, ventral prostatic tissue was collected and immediately homogenized (Polytron) in Trizol reagent (Life Technologies) at 1 ml per 50 mg tissue and stored at – 80°C for 3–5 days. Total RNA was isolated using Trizol reagent

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protocol and stored at – 80°C until Northern analysis. Northern analyses for TRPM2 and C3 mRNA were performed following low- and high-stringency washes; blots were sealed in plastic wrap, exposed to a phosphor screen for up to 2 h, and scanned for radioactivity emissions using a Phosphorimager 400S (Molecular Dynamics, Sunnyvale, CA). ImageQuant software, version 3.3, was used to correct for background and to quantify TRPM2 and C3 band densities.

In Vivo Study 4: Transgenerational Effects of Linuron and Di-n-butyl Phthalate (DBP) on the Histology of the Testis and Epididymis Treatments were administered in 2.5 ␮l of corn oil/g body weight by gavage to pregnant dams at 0, 100 mg linuron/kg/d (Linuron (Dupont lot 225, 100% Technical Grade) or 500 mg DBP/kg/d (DBP—Sigma Lot #109f-0386, purity of 99.8%). Dams in the control and DBP groups were dosed from gestational day 14 to day 3 of lactation, while in the linuron group, treatment was not continued after day 18 due to a reduction in maternal weight gain. The dose administered was adjusted daily, based on individual maternal weight changes throughout the dosing period. This phase of the experiment used 26 pregnant dams, randomly assigned (in a manner that provides equal means and distributions in maternal weight) to one of the treatment groups (control, n ⫽ 10; linuron and DBP, n ⫽ 8 dams). At 5 to 6 months-of-age, male rat offspring were necropsied (see Gray et al., 1999 for details) and organ weights were collected on 1–2 males randomly selected from each litter. The new data, presented herein, contain the results of the histopathological evaluation of the testicular and epididymal tissues. Tissues were placed in Bouin’s fixative for 24 h, then rinsed and stored in 70% alcohol, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by Experimental Pathology Laboratories, Inc. (Research Triangle Park, NC) for histopathological lesions.

Statistical Analyses In these studies, data were analyzed using PROC GLM, the SAS version 6.08, on the U.S. EPA IBM mainframe. TRPM2 and C3 data and the C3/ TRPM2 ratio were analyzed after log transformation due to heterogeneity of variance. The regression model for organ weights included body weight at necropsy as a covariate. Statistically significant effects (p ⬍ 0.05, F/t statistic) were examined using the LSMEANS procedure on SAS (t-test) to compare the control (castrate plus TP group) to the treated groups, t-tests being appropriate for a priori hypothesis. We hypothesized that linuron, vinclozolin, and flutamide would reduce androgen-dependent tissue weights in vivo, while in vitro, linuron would compete with R1881 for AR in prostatic cytosol, COS cells, and reduce DHT-induced gene expression in CV-1 cells. In addition, we expected linuron to reduce the C3/TRPM2 ratio by both increasing TRPM2 and decreasing C3 mRNA levels. Reported ED 50 values from the in vitro studies were estimated from the dose-response curves.

RESULTS

In Vitro Binding Assays In COS cells transiently transfected with the hAR, linuron inhibited R1881 AR binding by about 25% at 5 ␮M (p ⬍ 0.03), with an EC 50 of about 20 ␮M in this assay (Fig. 1A). Linuron competed with 10 ␮M [3H] R1881 for AR binding with an EC 50 of about 200 ␮M in a competitive binding assay using homogenized rat prostate (Fig. 1B).

Inhibition of DHT-Induced Transcriptional Activation in CV-1 and MDA-MB-453-KB2 Cells In CV-1 cells transiently co-transfected with the hAR and MMTV-luciferase reporter, linuron inhibited 0.1 nM DHTinduced transcriptional activation at doses as low as 10 ␮M (p ⬍ 02), which also was the EC 50 value (Fig. 1C). In the MDA-MB-453-KB2, linuron-inhibited DHT-induced luciferase expression significantly at 5 ␮M and above with an EC 50 of about 10 ␮M (Fig. 1D). Examination of the effects of linuron on luciferase expression in the CV-1 cell co-transfected with CMV-ABC, which is constitutively active, and 5 ␮g MMTV-luciferase revealed that linuron treatment at these concentrations was not cytotoxic, since luciferase expression was not reduced (data not shown). In Vivo Study 1: Hershberger Assay In the castrate-immature, TP-treated male rat, administration of linuron or vinclozolin for 7 days significantly reduced androgen-dependent seminal vesicle, ventral prostate gland, and levator ani plus bulbocavernosus muscle (LABC) weights (Table 1). Body, pituitary, liver, and kidney weights were unaffected, while adrenal weight was significantly increased by linuron and vinclozolin treatments (Table 1). Adult Studies 2 and 3: Adult Castrate plus T-treated Response to Linuron Treatment at 100 mg/kg/day—Experiments with 7 or 4 Days of Dosing When treatment was administered for 7 days, linuron reduced ventral prostate, epididymal, and LABC weights; however, seminal vesicle weight was unaffected (Table 2). Flutamide, which is a more potent AR-antagonist than linuron in vitro and in vivo, had a greater effect than linuron on the seminal vesicle, ventral prostate, and epididymis, while in contrast, linuron was as effective as flutamide in reducing LABC weights to nearly castrate-no-TP control levels, indicating a complete inhibition of androgen action. Linuron and flutamide treatments reduced body weight gain, while flutamide increased liver weight (Table 2). Neither treatment reduced serum testosterone levels. In the 4-day experiment, flutamide and linuron treatments de-repressed (increased) TRPM2 and decreased C3 mRNA levels. The ratio of C3 to TRPM2 (log transformed to correct for heterogeneity of variance, the standard deviation being proportional to the mean) was significantly decreased by both flutamide (p ⬍ 0.02) and linuron (p ⬍ 0.05) treatments (Fig. 2). The TRPM2 increase, produced by linuron treatment, was marginally significant (p ⬍ 0.04 one-tailed t-test), while the decrease in C3 was not significantly reduced. Flutamide reduced sex accessory gland and levator ani plus bulbocavernosus weights, while linuron did not (Table 3).

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FIG. 1. In vitro effects of linuron. Linuron (LIN) competitively inhibits R1881 binding to the human (A) and rat (B) androgen receptors in whole cell and cell-free binding assays, respectively. For comparative purposes, the inhibition of R1181 binding to hAR by DHT is displayed in A, while B displays binding data for rat AR by hydroxyflutamide (OH-FLUT). The data in C and D demonstrate that linuron displays antiandrogenic activity, antagonizing 0.1 nM DHT-induced gene (luciferase) expression in CV-1 (Fig. 2C) cells and MDA-453-KB2 (Fig. 2D) cells (Asterisks denote p ⬍ 0.01).

Study 4: Histological Examination of Reproductive Tissues of Male Offspring, Exposed in Utero to Linuron and DBP Epididymal and testicular malformations were noted in the linuron and DBP groups (Fig. 3), with more than 50% of the males exposed to either linuron or DBP displaying agenesis or atrophy of one or both organs. Histological examination of the tissues by light microscopy revealed lesions in the testis and epididymides of linuron- and DBP-treated male rat offspring. The tissues of DBP-treated male offspring were similarly affected. In the testes of the linuron group, histological evaluation by light microscopy revealed that 62% (8/13) displayed seminiferous tubules devoid of active spermatogenesis, with a severity grade of “moderately severe” (4 on a scale of 0 to 5) or “severe/high” (a score of 5, the worst case lesion) while none of the 15 control animals examined displayed any testic-

ular lesions (Fig. 3). In the epididymis of the 15 control males, the only anomaly, present in 4/15, was minimal focal mononuclear cell infiltration in the interstitium (severity, 1/minimal for all 4). Rats in the linuron group displayed a variety of epididymal lesions, including, diffuse epithelial hypertrophy (50% with mild to moderate severity; see Fig. 3) and hypospermia (58% with a severity grade of “moderate to severe”). DBP-treated males displayed nearly identical histological alterations of the testis and epididymis. The epididymal lesions included diffuse epithelial hypertrophy (63% with mild to moderate severity), and hypospermia (69% with a severity grade of “moderate to severe”). In the DBP group, 50% displayed seminiferous tubules that lacked active spermatogenesis.

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TABLE 1 Effects of Linuron (100 mg/kg/day orally) or Vinclozolin (200 mg/kg/day orally) for Seven Days on Organ Weights in Immature Castrate-TP (50 mg/day or Oil-Treated sc) Male SD Rats Vehicle, n ⫽ 5 (no TP)

Control, n ⫽ 7 (50 ␮g TP/day)

Linuron, n ⫽ 5 (50 ␮g TP/day)

Vinclozolin, n ⫽ 5 (50 ␮g TP/day)

167 ⫾ 6.4 8.9 ⫾ 0.37 7.44 ⫾ 0.43 42.3 ⫾ 5.9 a 1.52 ⫾ 0.05 54.2 ⫾ 3.8 18.5 ⫾ 1.8 b 9.9 ⫾ 1.9 b

171 ⫾ 5.7 8.4 ⫾ 0.43 8.06 ⫾ 0.34 32.1 ⫾ 1.3 1.60 ⫾ 0.06 59.0 ⫾ 2.8 91.8 ⫾ 5.5 63.2 ⫾ 3.6

161.3 ⫾ 5.1 7.9 ⫾ 0.25 8.02 ⫾ 0.35 44.6 ⫾ 4.1 b 1.48 ⫾ o.04 49.3 ⫾ 2.9 a 51.5 ⫾ 3.8 b 40.0 ⫾ 2.5 a

161.0 ⫾ 4.7 10.2 ⫾ 0.81 8.47 ⫾ 0.29 47.3 ⫾ 2.5 b 1.45 ⫾ 0.06 48.5 ⫾ 2.5 a 30.5 ⫾ 1.6 b 19.9 ⫾ 3.3 b

170 ⫾ 6.2 b

127 ⫾ 5.9 b

Body weight (g) Pituitary (mg) Liver (g) Adrenals (mg) Kidneys (g) Body-weight gain during dosing (g) Seminal vesicle (mg) Ventral prostate (mg) Levator ani plus bulbocavernosus (mg)

89.3 ⫾ 4.8 b

217.5 ⫾ 5.5

Note. TP ⫽ testosterone propionate. a p ⬍ 0.05. b p ⬍ 0.01.

DISCUSSION

The current study demonstrates that linuron is an androgen receptor antagonist in vitro and in vivo. Linuron competed with a synthetic androgen for binding to human AR, with EC 50 of about 20 ␮M, and to rat AR (isolated from prostatic tissue) with an apparent EC 50 of about 200 ␮M. In in vitro studies, linuron inhibited DHT-induced gene expression in 2 different cell assays (CV-1 and MDA-MB-453) with an EC 50 of about 10 ␮M, while in in vivo studies, linuron altered the expression of the androgen-regulated genes as indicated by a significant decrease in the C3/TRPM2 ratio. Linuron also inhibited TPinduced growth of the ventral prostatic, seminal vesicular, and levator ani/bulbocavernosus tissues in the immature castrate male rat model. An analysis of TRPM2 mRNA levels allows

for an in vivo confirmation of the antiandrogenic mechanism of action seen in vitro (Bettuzzi et al, 1992). TRPM2 levels are increased for the first few days after prostatic regression has been initiated in the adult male rat. Kelce et al., (1997) demonstrated that exposure to known antiandrogens flutamide, vinclozolin, and p,p⬘-DDE resulted in significant changes in the expression of TRPM2 mRNA. Taken together, these data provide an unequivocal pattern of antagonism of AR by linuron. For our short-term (4 –7 day), in vivo studies, we selected a dosage level that produced a high incidence of malformations in male rat offspring when administered to the dam on days 14 –18 of pregnancy. The Hershberger assay, which uses castrated, immature, androgen-treated rats, has been used for

TABLE 2 Seven Days of Treatment of Adult Castrate-T (2.5-cm silastic implant) SD Rats with Linuron (100 mg/kg/day orally) and Positive Control Flutamide (100 mg/kg/day orally)

Body weight (g) Weight gain (g) Epididymis (mg) Ventral prostate (mg) Seminal vesicle (mg) Levator ani plus bulbocavernosus (mg) Liver (g) Number of animals Serum T ng/ml Note. T ⫽ testosterone. a p ⬍ 0.05 vs. control (castrate ⫹ T). b p ⬍ 0.01 vs. control (castrate ⫹ T).

Castrate (no T)

Control (castrate ⫹ T)

Flutamide (100 mg/kg/day ⫹ T)

Linuron (100 mg/kg/day ⫹ T)

412 a ⫾ 9.2 37.9 ⫾ 7.4 207 b ⫾ 15 74.5 b ⫾ 9.9 348 b ⫾ 18

444 ⫾ 7.3 35.7 ⫾ 4.2 439 ⫾ 20 548 ⫾ 44 1494 ⫾ 101

416 b ⫾ 5.7 5.4 b ⫾ 5.3 258 b ⫾ 13 128 b ⫾ 9.7 516 b ⫾ 53

404 a ⫾ 5.3 5.0 b ⫾ 3.6 366 b ⫾ 20 367 b ⫾ 35 1453 ⫾ 138

822 b ⫾ 35 14.1 a ⫾ 0.34 8 0b

1142 ⫾ 70 15.9 ⫾ 0.81 7 1.40 ⫾ 0.12

881 b ⫾ 49 19.4 b ⫾ 0.80 5 1.28 ⫾ 0.07

865 b ⫾ 35 15.3 ⫾ 0.38 7 1.23 ⫾ .09

395


FIG. 2. Four days of treatment with linuron or flutamide (by gavage at 100 mg/kg/day) altered androgen-dependent gene expression in ventral prostatic tissue from adult-castrate-testosterone-treated male SD rats in a similar fashion, with flutamide being slightly more potent, in this regard. These treatments significantly reduced the C3/TRPM2 ratios as compared to the vehicle (corn oil)-treated, castrate-testosterone-treated control group.

decades to detect antiandrogenic chemicals (Hershberger, 1953). The linkage by dose of the short-term mechanistic studies with our developmental study of linuron demonstrates that small, but significant changes in organ weights and in situ gene expression in “screening” assays should be cause for concern because similarly exposed fetal males are malformed. Although several mechanisms of action could account for the effects of linuron in our in vivo studies, both in vitro and in vivo data provide evidence that linuron is acting as an AR antagonist. In the castrate-androgen-treated model, treatment with chemicals that act as AR ligands, lower tissue AR levels, inhibit 5␣ reductase, or stimulate testosterone metabolism can reduce DHT-dependent ventral prostate and seminal vesicle

weights. In contrast, 5␣ reductase inhibitors do not inhibit androgen-dependent LABC growth. In our studies, the fact that linuron-treatment for 7 days in the androgen-treated castrate male rat reduced both LABC and ventral prostate weights without reducing serum levels, is consistent with the hypothesis that linuron prevents testosterone-induced growth of these tissues by acting as an AR antagonist. In contrast to the effects of linuron-treatment for 7 days on the androgen-dependent tissue weights, treatment for 5 days was without effect, although treatment for this duration with the more potent AR antagonist flutamide was effective. Our results with linuron in the Hershberger assay are consistent with several early reports indicating that the castrate-immature male rat is more sensitive than is the adult-castrate (Fig. 4). Hooker (1942) reported that the accessory glands are most sensitive at 40 – 60 days-of-age when they are undergoing their most rapid development. These data indicate that inhibition of androgen-dependent growth of immature sex accessory tissues is more sensitive to disruption than initiation of tissue regression and apoptosis in young adult, sexually mature animals. The lack of sensitivity of the mature animal arises, in part, from the fact that some tissues have relatively low AR levels and require a low level of androgens for maintenance, and complete regression of the tissue in the absence of an androgen can take several weeks. In contrast, in the immature male rat, AR levels are relatively high and the growth rate of these tissues from 40 to 60 days-of-age is remarkable (Monosson et al., 1999; Stoker et al., 2000). In addition, 7 days of dosing, as compared to 4 or 5 days, also enhances the sensitivity of these assays (Ashby et al., 2000). For these reasons, we recommend that when one is screening chemicals that may be relatively weak antiandrogens, castrateimmature animals should be dosed at least 7 days. This

TABLE 3 TRPM2 Results for Adult Castrate-T (2.5-cm silastic implant) SD rats Treated with Linuron (100 mg/kg/day orally) and Positive Control Flutamide (100 mg/kg/day orally) for 4 Days

Body weight Serum T (ng/ml) Ventral prostate Seminal vesicle Epididymis Liver LABC muscles Optical density units: TRPM2 C3

Castrate (no T)

Control (castrate ⫹ T)

Flutamide (100 mg/kg/day ⫹ T)

Linuron (100 mg/kg/day ⫹ T)

408 ⫾ 5.8 0.1 242 ⫾ 21 818 ⫾ 91 396 ⫾ 23 12.4 ⫾ 0.72 1060 ⫾ 30

40 ⫾ 9.18 1.9 666 ⫾ 62 1966 ⫾ 116 522 ⫾ 11 13.2 ⫾ 0.22 1354 ⫾ 71

396 ⫾ 8.6 2.1 456 a ⫾ 66 863 b ⫾ 49 385 b ⫾ 33 13.8 ⫾ 0.34 1098 b ⫾ 85

398 ⫾ 9.1 2.2 713 ⫾ 83 2128 ⫾ 140 514 ⫾ 19 12.8 ⫾ 0.38 1252 ⫾ 30

33,498 ⫾ 7716 263 ⫾ 110

786 ⫾ 269 16,718 ⫾ 3138

9593 b ⫾ 2756 12,382 ⫾ 2634

3914 c ⫾ 1640 10,178 ⫾ 1657

p ⬍ 0.05 vs. control (castrate ⫹ T). p ⬍ 0.01 by a 2-tailed t-test vs. control (castrate ⫹ T). c p ⬍ 0.04 by a 1-tailed test vs. control (castrate ⫹ T). a b

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approach closely resembles the standard methods for assessing antiandrogens adopted by official organizations in 1962 (Dorfmann), the EDSTAC Final Report (1999), and the Hershberger assay protocol currently under consideration by the OECD. When linuron is administered from GD 14 –18 at the same dosage level used in the Hershberger assay (100 mg/kg/d), the male offspring display a high incidence of testicular and epididymal abnormalities. As previously reported in male offspring, anogenital distance, adjusted by analysis of covariance for body weight, was reduced by about 30% and the incidence of areolas was increased from 0% in controls to more than 44% by in utero linuron-treatment. At necropsy, one male of 13 from the linuron group displayed epispadias (partial hypospadias, with the urethral opening being half way down the phallus) (Fig. 3) and androgen-dependent tissues were permanently reduced in size, including the seminal vesicles, ventral prostates, levator ani/bulbocavernosus muscles, and the epididymides. In addition, testis weight also was reduced by in utero linuron exposure. Although linuron is clearly an AR antagonist in vitro and in the castrate-hormone-treated animal model, the profile of in utero effects more closely resembles one produced with in utero phthalate ester treatment than with AR antagonist. For example, when DBP is administered at 500 mg/kg/day from day 14 of pregnancy to the third day of lactation, 46% of the males display epididymal and testicular agenesis while only 6.2 % display hypospadias (Gray et al., 1999b). In contrast, perinatal treatment with an AR antagonist like procymidone or vinclozolin has much less effect on the epididymides and testes at dosage levels that induce hypospadias in 100% of the males. The differential response to the T- and DHT-dependent tissues to AR antagonists in the rat has been noted for flutamide by other investigators (Imperato-McGinley et al., 1992) in addition to our observations with procymidone and vinclozolin. In this regard, it is puzzling that the developmental effects of linuron resemble the effects produced by treatment with DBP or DEHP as opposed to the profile of malformations induced by vinclozolin or other AR antagonists. It is possible that linuron, like DEHP or DBP, inhibits fetal steroid production. Several laboratories, including our own, are investigating whether linuron alters fetal T synthesis or displays additional

397

FIG. 4. Effects of linuron on the ventral prostate, seminal vesicle plus coagulating glands with fluids and levator ani plus bulbocavernosus muscle weights administered by gavage at 100 mg/kg/day in 3 assays, expressed as percent of the androgen-treated control group. The three in vivo studies are: (1) the 7-day Hershberger assay using castrate-immature, testosterone propionatetreated SD rats (the Hershberger assay), and castrate-adult-testosterone implanted SD rats treated for (2) 7 days or (3) 4 days. This figure shows that when treated for 7 days, the androgen-dependent tissues of the immature animal are more affected by linuron than those of the adult animal. These data also show that, although optimal for in vivo gene expression, 4 days of treatment with linuron is not of sufficient duration to detect its antiandrogenic effects on these ventral prostate, seminal vesicle, and levator ani plus bulbocavernosus muscle weights. Asterisks indicate that the value differs from control by p ⬍ 0.01).

mechanisms of antiandrogen action in the fetal male rat (B. S. McIntyre and L. G. Parks laboratories, personal communications). While the dosage level of linuron used in the current investigation is above the current NOAEL for this herbicide, it is of concern that developmental toxicology and multigenerational studies failed to identify that this pesticide produces malformations of the reproductive tract and other androgen dependent tissues. When linuron was administered over 3 generations, no reproductive malformations were noted and 125 ppm (dietary) was identified as a NOAEL (Hodge et al., 1968). Khera et al. (1978) reported that linuron was not teratogenic in a standard developmental toxicity test using the rat, at dosage levels up to

FIG. 3. Linuron-induced developmental alterations in male rat offspring. Exposing the dam on gestational days 14 –18 to 100 mg linuron/kg/d produces malformations of the male reproductive tract. Upper left panel: Adult control male rat glans penis. Upper right panel: Linuron-treated male rat displaying epispadias. This was the only (1/13 examined) male with malformed external genitalia. Second row, left panel: Testis and epididymis with epididymal fat pad of an adult control male rat. Second row, right: Fluid-filled, flaccid testis (with severe seminiferous tubular atrophy), with agenesis of the caput epididymis from a male rat in linuron group. This testis weighed about 1.06 g versus control testes weights which average 1.8 g. Third row, left panel: Entire epididymis from adult control male rat with testis and epididymal fat removed. The cauda is to the right and the caput epididymis is at the left. Third row, right panel: Linuron-treated male rat cauda and corpus epididymis with agenesis of the caput epididymis. Fourth row, left panel: H&E-stained histological cross section of the testis of an adult control male rat. Fourth row, right panel: Moderate seminiferous tubular atrophy of the testis of a male rat exposed in utero to linuron. Bottom row, left panel: H&E-stained histological section of the cauda epididymis of a control male rat with sperm in the lumen. Bottom row, right panel: Cross section of the epididymis of a linuron-treated rat displaying epithelial hyperplasia.

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100 mg/kg/day. Based on the results of the multigenerational study from 1968, linuron has been described as a “nonspecific systemic” toxicant, which would lead one to conclude that linuron is not an “endocrine disrupter.” In fact, the retardation of growth seen by Hodge et al. (1968) and Cook et al., (1993) likely is related to the ability of linuron to disrupt thyroid and the neuroendocrine axes, lowering serum T4 and inducing overt neurotoxicity (salivation, lacrimation, urination, and lethargy after an acute dose of 200 mg/kg/day accompanied by altered brain neurotransmitter levels; Rehnberg, 1988), rather than being a “nonspecific” toxicant. Recently, McIntyre et al. (1999) repeated and extended our observations on the developmental reproductive toxicity of linuron in the rat. When they dosed pregnant rats with linuron during pregnancy (days 10 – 21) they observed a dose-related incidence of reproductive malformations in about 4% of the male offspring affected at 12.5 mg/kg/day, the lowest dose tested. It is clear that these malformations would not be evident in a standard teratology study with dosing from GD 6 –15, as this dosing regime does not include the period of sexual differentiation. Even if dosing were extended to day 19 of pregnancy, a teratology study would be unlikely to detect these reproductive malformations if only fetal animals were examined. With regard to the older multigenerational study (Hodge, 1968), studies of that era did not typically include an assessment of AGD, androgen-dependent tissue weights, testis or epididymal sperm numbers, or areolas and nipples in male offspring. In addition, using a sample size of 20, these studies would be unable to statistically detect malformations in the F1 if only 4% of the population was affected. In fact, about 25% of the animals would have to be malformed to attain statistical significance in only 20 F1 animals. Although, the new EPA Harmonized Multigenerational Reproduction Test requires that, in addition to the 20 F1 animals per sex per dose continued on study, 3 rats per sex per litter are examined for gross malformations at weaning, it is evident that some of malformations cannot be identified in immature animals. For this reason, transgenerational studies (i.e., EDSTAC Final Report (EPA, 1998); Gray et al., 1999a,b; Mylchreest et al., 1998, 1999; Ostby et al., 1999) now include an assessment of all F1 pups through puberty or even later in life. In summary, we report here that linuron is an AR antagonist with sufficient potency to produce effects that are clearly evident in vivo. Linuron competes with androgens for AR binding. It inhibits androgen-induced gene expression in vitro and short-term linuron treatment reduces the size of androgendependent tissues in vivo in a manner indicative of its ability to act as an AR antagonist. We suspect that this mechanism contributes to the malformations of androgen-dependent tissues when linuron is administered in utero. However, based on the fact that the profile of malformations is unusual for an AR antagonist, we suspect that linuron may display additional mechanisms of action.

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LINURON: ANDROGEN RECEPTOR ANTAGONIST? Khera, K. S., Whalen, C., and Trivett, G. (1978). Teratogenicity studies on linuron, malathion, and methoxychlor in rats. Toxicol. Appl. Pharmacol. 45, 435– 444. McIntyre, B. S., Barlow, N. J., Wallace, D. G., and Foster, P. M. (1999). Effects of the antiandrogen linuron on androgen-dependent reproductive development in the male CD rat. European Teratology Society Abstracts P14. Monosson, E., Kelce, W. R., Lambright, C., Ostby, J., and Gray, L. E., Jr. (1999). Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat. Toxicol. Ind. Health 15, 65–79. Mylchreest, E., Cattley, R., and Foster, P. M. D. (1998). Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl) phthalate: An antiandrogenic mechanism. Toxicol. Sci. 43, 47– 60. Mylchreest, E., Sar, M., Cattley, R. C., and Foster, P. M. (1999). Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide. Toxicol. Appl. Pharmacol. 1556, 81–95. Ostby, J., Kelce, W. R., Lambright, C., Wolf, C. J., Mann, P., and Gray, L. E., Jr. (1999). The fungicide procymidone alters sexual differentiation in the

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male rat by acting as an androgen-receptor antagonist in vivo and in vitro. Toxicol. Ind. Health 15, 80 –93. Parks, L. G., Ostby, J. S., Lambright, C. R., Abbott, B. D., and Gray, L. E., Jr. (1999). Perinatal BBP and DEHP exposures induce antiandrogenic effects in SD rats. Biology of Reproduction 606, 153, A192. Rehnberg, G. L., Goldman, J. M., Cooper, R. L., Gray, L. E., Jr., Hein, J. F., McElroy, W. K., and Booth, K. C. (1988). Effect of linuron on the brainpituitary-testicular reproductive axis in the rat. Toxicologist 8, 121. Schardein, J. L. (1993). Hormones and hormonal antagonists. In Chemically Induced Birth Defects (J. L. Schardein, Ed.), pp. 271–339. Marcel Dekker, New York. Stoker, T, Parks, L., Gray, L. E., Jr., and Cooper, R. (2000). Endocrinedisrupting chemicals: Prepubertal exposures and effects on sexual maturation and thyroid function in the male rat. A focus on the EDSTAC recommendations. Endocrine Disrupter Screening and Testing Advisory Committee. Crit. Rev. Toxicol. 30, 197–252. Waller, C., Juma, B., Gray, L. E., Jr., and Kelce, W. R. (1996). Threedimensional quantitative structure-activity relationships for androgen receptor ligands. Toxicol. Appl. Pharmacol. 137, 219 –227. Wong, C., Kelce, W. R., Sar, M., and Wilson, E. M. (1995). Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J. Chem. Biol. 270, 19998 –20003.