Verification of responses of Japanese medaka (Oryzias latipes) to anti‐androgens,vinclozolin and flutamide,in short‐term assays

Various testing methods for the detection of the endocrine disruptive activities of chemicals have been developed in freshwater fish species. However, a few relatively easier specific methods for detecting anti‐androgenic activities are available for fish. The aim of this study was to verify the papillary process in Japanese medaka (Oryzias latipes) as an indicator of the anti‐androgenic activity of chemicals. Japanese medaka were exposed to two types of anti‐androgenic compounds, vinclozolin and flutamide, using two short‐term assays; one was conformed to the existing short‐term reproduction assay using adult fish (adult test) and the other was a test based on the same methods but using juvenile fish at the beginning of exposure (juvenile test). Significant decreases in male papillary processes were observed in the juvenile test treated with the highest concentration of both antiandrogens (640 µg l^–1 vinclozolin and 1000 µg l^–1 flutamide); however, no significant effects were observed in the adult test. Consequently, our results indicate that papillary processes in Japanese medaka can be used as the end‐point for screening the anti‐androgenic activity of chemicals using juvenile fish for a specific period based on the existing short‐term reproduction assay. Copyright © 2013 John Wiley & Sons, Ltd.     

农利灵对斑马鱼胚胎发育的毒性

[目的]探讨农用杀菌剂农利灵（vinclozolin）对斑马鱼（Brachydanio rerio）的胚胎发育毒性及其机制。[方法]采用水浴染毒法，将受精后2h（hours post fertilization，hpf）内的斑马鱼卵分别暴露于浓度为12．5、25、50、100μg／mL的农利灵，作为4个染毒组，同时设置溶剂（DMSO）对照组、10μg／mL睾酮（testosterone）组及10μg／mL睾酮＋50μg／mL农利灵混合组。通过死亡率、孵化时间、发育畸形等指标评价农利灵的胚胎发育毒性。[结果]农利灵染毒组的胚胎死亡率分别为52．6％、68．4％、85．0％、100．0％，明显高于溶剂对照组（10．0％），P〈0．01，并呈剂量-效应关系。比较孵化时间，12．5μg／mL农利灵染毒组高于溶剂对照组（t=4．077，P〈0．05）。 10μg／mL睾酮＋50μg／mL农利灵混合组的死亡率为51．2％，低于50μg/mL农利灵染毒组的死亡率85．0％（X^2=6．53，P〈0．05）。此外，在农利灵染毒组观察到脊柱弯曲、心包囊肿等发育畸形，其畸形与睾酮染毒组所观察到的畸形相似。[结论]农利灵可延迟斑马鱼的胚胎发育、具有胚胎致死毒性和致畸毒性，其毒性至少有部分是来源于抗雄性激素样作用。     

The effect of a prenatal androgen disruptor, vinclozolin, on gubernacular migration and testicular descent in rats

Background/purpose

Androgen has been shown to regulate inguinoscrotal testicular descent. This study aims to clarify the effect of one of the major endocrine disrupters, vinclozolin (V), on both gubernacular migration and inguinoscrotal testicular descent in rats.

Methods

Time-pregnant rats were segregated into 2 groups. In group I, the rats were administered 200 mg/kg/d of V by gavage on days 15 to 18 of gestation. In group II, the rats were administered the same volume of solvent and were used as controls. At birth, the anogenital distance was measured in pups, and gubernacular migration was examined at 10 days of age in some of male offspring. Next, the incidence of testicular descent and the growth of external genitalia were investigated in the remaining male offspring at 60 days of age. The χ² test was used for statistical analysis of the results.

Results

At birth, the anogenital distance (AGD) index decreased significantly more in group I than in group II in male offspring. However, there was no significant difference in the AGD index between the 2 groups in the female offspring. At 10 days of age, an aberrant migration of the gubernaculum was found in the 51.5% of V-treated rats in group I. At 60 days of age, the incidence of cryptorchidism was 57.7% in group I and 0% in group II (P < .05). In addition, hypospadias with cleft phallus and pseudo vagina with a blind pouch also were observed in some of the V-treated rats.

Conclusions

Prenatal administration with V thus caused intrauterine defects, which resulted in testicular maldescent caused by the induction of an aberrant migration of the gubernaculum associated with an abnormal extension of the processus vaginalis, and this may have been caused by the antiandrogenic effect of V in utero.     

A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists.

The U.S. Environmental Protection Agency has proposed that in vitro assays for estrogen receptor (ER)- and androgen receptor (AR)-mediated actions be included in a Tier-I screening battery to detect hormonally active chemicals. Herein we describe the development of a novel stable cell line, MDA-kb2, for screening of androgen agonist and antagonists and to characterize its specificity and sensitivity to endocrine-disrupting chemicals. The breast cancer cell line, MDA-MB-453, was stably transformed with the MMTV.luciferase.neo reporter gene construct. Since both GR and AR are present in the MDA-MB-453 cells, and both receptors can act through the MMTV promoter, compounds that act through either AR or GR activate the MMTV luciferase reporter. As expected, AR agonists such as dihydrotestosterone (DHT), and GR agonists such as dexamethasone (DEX), corticosterone, and aldosterone induce luciferase expression at appropriate concentrations. DHT consistently produced 3-9-fold induction at concentrations from 0.1 to 10 nM. At 1 to 1000 nM, DEX induced luciferase activity 1.3-19.5-fold. To distinguish AR- from GR-mediated ligands, chemicals were assayed concurrently with the antiandrogen, hydroxyflutamide (OHF), which blocks AR- but not GR-mediated responses. In addition, known AR antagonists, including hydroxyflutamide, vinclozolin, vinclozolin metabolites M1 and M2, p,p'-DDE, and linuron inhibited DHT-induced luciferase gene expression at appropriate concentrations in this system. We have found that these cells are relatively easy to culture and maintain. Responsiveness was monitored over time and was stable for more than 80 passages. Some advantages of this assay are that it is relatively rapid (2 days), eliminates the need for transfection, can be conducted in a 96-well plate format, and produces consistent reproducible results. In summary, we have developed a cell line that can be used to screen chemicals, not just for AR- but for GR-mediated activities as well.     

Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat

The incidence of hypospadias is increasing in young boys, but it remains unclear whether human exposure to endocrine disrupting chemicals plays a role. Risk assessment is based on estimation of no-observed-adverse-effect levels for single compounds, although humans are exposed to combinations of several anti-androgenic chemicals. In a mixture (MIX) study with three androgen receptor antagonists, vinclozolin, flutamide and procymidone, rats were gavaged during gestation and lactation with several doses of a MIX of the three chemicals or the chemicals alone. External malformations of the male reproductive organs were assessed on PND 47 using a score from 0 to 3 (normal to marked) for hypospadias. Markedly increased frequencies were observed after exposure to a MIX of the three chemicals compared to administration of the three chemicals alone. Anogenital distance at PND 1, nipple retention at PND 13, and dysgenesis score at PND 16 were highly correlated with the occurrence of hypospadias, and MIX effects were seen at doses where each of the individual chemicals caused no observable effects. Therefore, the results indicate that doses of anti-androgens, which appear to induce no hypospadias when judged on their own, may induce a very high frequency of hypospadias when they interact in concert with other anti-androgens.     

Dysgenesis and Histological Changes of Genitals and Perturbations of Gene Expression in Male Rats after In Utero Exposure to Antiandrogen Mixtures

We investigated the ability of a mixture of three androgen receptorantagonists to induce disruption of male sexual differentiationafter perinatal exposure. The aim was to assess whether thejoint effects of vinclozolin, flutamide, and procymidone canbe predicted based on dose-response data of the individual chemicals.Chemicals were administered orally to pregnant Wistar rats fromgestational day 7 to postnatal day 16. Changes in reproductiveorgan weights and of androgen-regulated gene expression in prostatesfrom male rat pups were chosen as end points for extensive dose-responsestudies. With all end points, the joint effects of the threeantiandrogens were dose additive. Histological evaluations showedthat dysgenesis and hypoplasia of prostates, seminal vesicles,and epididymis were seen with the highest mixture doses. Nochanges were observed in any single-compound low-dose groupfor these lesions, nor were there histopathological changesin the testes. Pronounced dysgenesis of external genitals wasobserved with all doses of the mixture, and severe dysgenesiswas seen with a mixture for which the individual compounds causedno effects. A combination of doses of each chemical that onits own did not produce significant reductions in the weightsof seminal vesicles and PBP C3 expression induced a marked mixtureeffect. Thus, antiandrogens cause additive effects on end pointsof various molecular complexities such as alterations at themorphological and the molecular level. Exposure to antiandrogens,which appears to exert only small effects when judged on a chemical-by-chemicalbasis, may induce marked responses in concert with, possiblyunrecognized, similarly acting chemicals.     

Characterization of the period of sensitivity of fetal male sexual development to vinclozolin.

Vinclozolin is a fungicide whose metabolites are androgen receptor (AR) antagonists. Previous work in our laboratory showed that perinatal administration of vinclozolin to rats results in malformations of the external genitalia, permanent nipples, reduced anogenital distance (AGD), and reduced seminal vesicle, ventral prostate, and epididymal weights. The objectives of this study were to determine the most sensitive period of fetal development to antiandrogenic effects of vinclozolin and to identify a dosing regime that would induce malformations in all of the male offspring. Pregnant rats were dosed with 400 mg vinclozolin/kg/day on either GD 12-13, GD 14-15, GD 16-17, GD 18-19, or GD 20-21, or with corn oil (2.5 ml/kg) from GD 12 through GD 21 (Experiment 1). All 2-day periods in which significant effects were produced were included in an extended dosing period, GD 14 through GD 19, in which pregnant rats were dosed with 200 or 400 mg vinclozolin/kg (Experiment 2). In Experiment 1, significant effects of vinclozolin were observed in rats dosed on gestation days (GD) 14-15, GD 16-17, and GD 18-19, while the most significant effects were observed in rats treated on GD 16-17. These effects include reduced AGD; presence of areolas, nipples, and malformations of the phallus; and reduced levator ani/bulbocavernosus weight. In contrast, ventral prostate weight was reduced only in the GD 18-19 group. The expanded dosing regime (Experiment 2) increased the percentage of male offspring with genital malformations (> 92%), and retained nipples (100%), further reduced the weight of the ventral prostate, and reduced the weight of the seminal vesicles. In addition, malformations were more severe and included vaginal pouch and ectopic/undescended testes. The latter was induced only in the 400 mg/kg group. These data indicate that the reproductive system of the fetal male rat is most sensitive to antiandrogenic effects of vinclozolin on GD 16 and 17, although effects are more severe and 100 % of male offspring are affected with administration of vinclozolin from GD 14 through GD 19.     

Early-onset endocrine disruptor-induced prostatitis in the rat

BACKGROUND: Androgens are critical for specifying prostate development, with the fetal prostate sensitive to altered hormone levels and endocrine-disrupting chemicals (EDCs) that exhibit estrogenic or antiandrogenic properties. Prostatic inflammation (prostatitis) affects 9% of men of all ages, and > 90% of cases are of unknown etiology. OBJECTIVES: In this study we aimed to evaluate effects of in utero exposure to the antiandrogenic EDC vinclozolin, during the period of male reproductive tract development, on neonatal, prepubertal, and postpubertal prostate gland function of male offspring. METHODS: Fetal rats were exposed to vinclozolin (100 mg/kg body weight) or vehicle control (2.5 mL/kg body weight) in utero from gestational day 14 (GD14) to GD19 via oral administration to pregnant dams. Tissue analysis was carried out when male offspring were 0, 4, or 8 weeks of age. RESULTS: In utero exposure to vinclozolin was insufficient to perturb prostatic development and branching, although expression of androgen receptor and mesenchymal fibroblast growth factor-10 was down-regulated. Prostate histology remained normal until puberty, but 100% of animals displayed prostatitis postpubertally (56 days of age). Prostatic inflammation was associated with phosphorylation and nuclear translocation of nuclear factor-kappa B (NFkappaB) and postpubertal activation of proinflammatory NFkappaB-dependent genes, including the chemokine interleukin-8 and the cytokine transforming growth factor-beta1. Significantly, inflammation arising from vinclozolin exposure was not associated with the emergence of premalignant lesions, such as prostatic intra-epithelial neoplasia or proliferative inflammatory atrophy, and hence mimics nonbacterial early-onset prostatitis that commonly occurs in young men. CONCLUSIONS: These data are the first to unequivocally implicate EDCs as a causative factor and fill an important knowledge gap on the etiology of prostatitis.     

Diverse mechanisms of anti-androgen action: impact on male rat reproductive tract development

Scientists have identified environmental chemicals that display anti-androgenic activity via multiple mechanisms of action. Early studies focused on pesticides acting as androgen receptor (AR) antagonists but it soon became apparent that was not the only endocrine mode by which compounds affected the androgen signalling pathway. Classes of chemicals currently known to interfere with the androgen signalling pathway include dicarboximide fungicides (e.g. vinclozolin), organochlorine-based insecticides (e.g. p,p'-DDT and -DDE), conazole fungicides (e.g. prochloraz), plasticizers (phthalates) and urea-based herbicides (linuron). Phthalate esters (PEs) and vinclozolin appear to act primarily via a single mechanism of action, while others such as linuron and prochloraz, appear to display dual mechanisms of action. Exposure to PEs decreases mRNA expression of key steroidogenic enzymes and also the peptide hormone insulin-like peptide 3 (insl3) from the foetal Leydig cells. Hence, both androgen- and inls3-dependent tissues are affected. Vinclozolin and procymidone act solely through binding to the AR as antagonists thus blocking the action of androgen at the cellular level but do not affect foetal testosterone synthesis or insl3 gene expression. The compounds linuron and prochloraz are AR antagonists but also inhibit foetal testosterone synthesis, although unlike the PEs, mRNA expression of steroidogenic enzymes and insl3 are not affected. All the above chemicals disrupt androgen signalling in the foetal male rat and produce some malformations in common, but the precise profiles of effects in the offspring are pathognomonic for each mode of action. For example, the 'phthalate syndrome' vs. the 'vinclozolin syndrome' each displays a profile of effects which is clearly different. In summary, as more and more molecular studies with anti-androgenic compounds are conducted, the number of mechanisms by which compounds can affect the androgen signalling pathway is likely to increase. Furthermore, the effects of mixtures of these compounds are just beginning to be explored.     

Evaluation of a 15-day screening assay using intact male rats for identifying antiandrogens.

An in vivo screening assay using intact adult male rats has been evaluated for its ability to detect six antiandrogenic compounds via oral administration. The test compounds included cyproterone acetate (CPA), flutamide (FLUT), p,p'-DDE (DDE), di-n-butyl phthalate (DBP), linuron (LIN), and vinclozolin (VCZ). Two of the test compounds (DDE and FLUT) have been previously evaluated in the 15-day intact male assay with compound administration via intraperitoneal injection (ip). For the current studies, male rats were dosed for 15 days via oral gavage and euthanized on the morning of test day 15. The endpoints evaluated included final body and organ weights (liver, thyroid gland, testes, epididymides, prostate, seminal vesicles with fluid, accessory sex gland unit [ASG]), serum hormone concentrations (testosterone [T], estradiol [E2], dihydrotestosterone [DHT], luteinizing hormone [LH], follicle stimulating hormone [FSH], prolactin [PRL], T(3), T(4), and thyroid stimulating hormone[TSH]), and histopathology of the testis, epididymis, and thyroid gland; positive results for each endpoint are described below. In addition, an evaluation of immune system endpoints (humoral immune function, spleen and thymus weights, and spleen cell number) was conducted on a subset of animals dosed with either DDE or FLUT. All six endocrine-active compounds (EACs) increased relative liver weight. FLUT and VCZ caused the typical pattern for an androgen receptor (AR) antagonist, although not all endpoints were statistically significant for VCZ: decreased ASG weights, hormonal alterations (increased T, DHT, LH, and FSH), and induced Leydig cell hypertrophy and/or hyperplasia. CPA caused effects consistent with its mixed AR antagonist/progesterone receptor agonist activity: it decreased ASG weights, caused hormonal alterations (increased T and E2; decreased FSH), and caused spermatid retention. DBP, a compound with antiandrogen-like activity via a nonreceptor mediated mechanism, caused hormonal alterations (decreased T, DHT, and E2; increased LH, FSH, and PRL) and induced general testicular degeneration. LIN, a weak AR antagonist, decreased ASG weights, caused hormonal alterations (decreased T, DHT, and LH; increased E2), and caused spermatid retention. Unlike the other AR antagonists evaluated, DDE, a weak AR antagonist, did not alter reproductive parameters. All six antiandrogens caused some effects on thyroid parameters, although only CPA, DDE, and VCZ caused results consistent with a potential thyroid-modulator. FLUT and DDE did not alter the primary humoral immune response to SRBC, spleen or thymus weights, or spleen cell number. In the current study, 5 of the six test substances were identified as endocrine-active substances consistent with their known/proposed mechanism(s) of action. The effects that were observed in the current study via oral (gavage) compound administration were similar to the responses that were observed by the ip route in previous studies for DDE and FLUT. This report, in addition to the > 20 compounds that have already been examined using the 15-day intact male assay, supports this assay as a viable screening assay for detecting EACs, and also illustrates that the ability to identify EACs using the intact male assay will be equivalent regardless of the route of compound administration.