Widely used pesticides with previously unknown endocrine activity revealed as in vitro antiandrogens.

BACKGROUND: Evidence suggests that there is widespread decline in male reproductive health and that antiandrogenic pollutants may play a significant role. There is also a clear disparity between pesticide exposure and data on endocrine disruption, with most of the published literature focused on pesticides that are no longer registered for use in developed countries. OBJECTIVE: We used estimated human exposure data to select pesticides to test for antiandrogenic activity, focusing on highest use pesticides. METHODS: We used European databases to select 134 candidate pesticides based on highest exposure, followed by a filtering step according to known or predicted receptor-mediated antiandrogenic potency, based on a previously published quantitative structure-activity relationship (QSAR) model. In total, 37 pesticides were tested for in vitro androgen receptor (AR) antagonism. Of these, 14 were previously reported to be AR antagonists ("active"), 4 were predicted AR antagonists using the QSAR, 6 were predicted to not be AR antagonists ("inactive"), and 13 had unknown activity, which were "out of domain" and therefore could not be classified with the QSAR ("unknown"). RESULTS: All 14 pesticides with previous evidence of AR antagonism were confirmed as antiandrogenic in our assay, and 9 previously untested pesticides were identified as antiandrogenic (dimethomorph, fenhexamid, quinoxyfen, cyprodinil, λ-cyhalothrin, pyrimethanil, fludioxonil, azinphos-methyl, pirimiphos-methyl). In addition, we classified 7 compounds as androgenic. CONCLUSIONS: Due to estimated antiandrogenic potency, current use, estimated exposure, and lack of previous data, we strongly recommend that dimethomorph, fludioxonil, fenhexamid, imazalil, ortho-phenylphenol, and pirimiphos-methyl be tested for antiandrogenic effects in vivo. The lack of human biomonitoring data for environmentally relevant pesticides presents a barrier to current risk assessment of pesticides on humans.

The sensitivity of the MDA-kb2 cell in vitro assay in detecting anti-androgenic chemicals--identification of sources of variability and estimation of statistical power.

In vitro assays for anti-androgens have been developed as screening tools for the identification of androgen receptor (AR) antagonists. We explored the usefulness of such assays for experimental purposes that require quantitation of effects in a highly reproducible manner, such as multi-component mixture experiments or evaluation of extracts of complex environmental samples. We have investigated sources of experimental variation in the MDA-kb2 assay for AR-antagonists. By omitting phenol red from culture media, avoiding media changes and extending the period allowed for cell attachment, the dynamic range increased. Variations in luminescence readings decreased, with smaller coefficients of variation within- and between-experiments. Normalisation of luminescence values to positive controls improved experiment-to-experiment reproducibility and allowed pooling of data from independent experiments. We also performed statistical power analyses to determine the minimal suppression of androgenic (DHT) effects by test agents that are detectable as statistically significantly different from positive controls (so-called minimum significant differences, MSD). Using the modified assay protocol extensive concentration-response analyses were conducted with bisphenol A, BDE100 and vinclozolin. Our modified procedure improves considerably the reproducibility of the MDA-kb2 assay.

Evaluation of the rodent Hershberger bioassay on intact juvenile males--testing of coded chemicals and supplementary biochemical investigations.

Under the auspices of the Organization for Economic Cooperation and Development (OECD) the Hershberger assay on juvenile intact male rats is being validated as a screen for compounds with anti-androgenic potential. We participated in the testing of coded chemicals. Compounds included the positive control flutamide (FLUT, 3 mg/kg), linuron (LIN, 10, 100 mg/kg), p,p'-DDE (16, 160 mg/kg), and two negative substances, 4-nonylphenol (NP, 160 mg/kg) and 2,4-dinitrophenol (DNP, 10 mg/kg). Compounds were administered for 10 consecutive days by gavage to testosterone propionate (TP, 1 mg/kgs.c.)-supplemented rats. Uncoding revealed these results: compared to vehicle controls, treatment with TP resulted in increased androgen-sensitive tissue (AST) weights of ventral prostate (VP), seminal vesicles (SV), levator ani and bulbocavernosus muscles (LABC), Cowper's glands, and epididymides, and in decreased testes weight. When assessing anti-androgenic potential in TP-supplemented rats, FLUT decreased all AST weights, and increased testes weight. p,p'-DDE at the high dose, decreased final body weight and all AST weights, whereas the low dose only affected SV weight. LIN slightly decreased final body weight and decreased absolute SV and LABC and relative SV weights only at the high dose. NP decreased final body weight and only absolute SV weights, DNP was ineffective. Investigations not requested by OECD included measurement of liver enzymes and revealed strong induction of testosterone-metabolizing and phase II conjugating enzymes by p,p'-DDE. Our findings suggest that in principle the juvenile intact male rat can be used in the Hershberger assay to screen for anti-androgenic potential thereby contributing to a refinement of the assay in terms of animal welfare. However, in our hands this animal model was somewhat less sensitive than the peripubertal castrated rat. Final conclusions, however, can only be drawn on the basis of all available validation data. Results obtained with the negative reference compound NP suggest that a treatment-related decrement in body weights may affect AST weights and represent a confounding factor when screening for anti-androgenic properties. Finally, p,p'-DDE may affect AST weights by several mechanisms including enhanced testosterone metabolism.

[Progestagen use in oral contraception - basic knowledge]. / Grundlagen zur Gestagen-Komponente in der hormonalen Kontrazeption.

The effects of most prostagens used in hormone replacement therapy (HRT) and in hormonal contraception are not identical. They differ in their structure, the metabolisation, their bioavailability and their partial activities. Therefore, it does not make any sense to declare that the different progestagens with their different profiles belong to one class of substances, having all the same effect. In function of their partial activities, progestagens are used today specifically for HRT, for hormonal contraception or as an anti-androgen. Because of their metabolic profile, some newer progestagens can be considered to be specifically favourable, such as Norpregnanes, Dienogest and Drospirenone. This review summarizes the properties and partial activities of progestagens used today and analyzes its clinical consequences.

Evaluation of the rodent Hershberger bioassay: testing of coded chemicals and supplementary molecular-biological and biochemical investigations.

Under the auspices of the Organization for Economic Cooperation and Development (OECD) the Hershberger assay is being validated as an in vivo screen for compounds with (anti)androgenic potential. We participated in the final activity, the testing of coded chemicals. Test compounds included trenbolone (TREN; 1.5, 40 mg/kg), testosterone propionate (TP; 0.4 mg/kg), flutamide (FLUT; 3mg/kg), linuron (LIN; 10, 100mg/kg), 1,1-bis-(4-chlorophenyl)-2,2-dichloroethylene (p,p'-DDE; 16, 160 mg/kg), and two negative reference substances, i.e., compounds not considered to affect androgen-sensitive tissue weights (ASTWs) in the Hershberger assay, namely 4-nonylphenol (NP; 160 mg/kg) and 2,4-dinitrophenol (DNP; 10mg/kg); TREN, LIN, p,p'-DDE, NP, and DNP being used under code. Compounds were administered for 10 days by oral intubation or subcutaneous injection (TP). Additional investigations not mandatorily requested by OECD included organ gravimetry of the liver, gene expression analysis in prostate using quantitative RT PCR for prostate specific binding protein polypeptide C3 (PBPC3) and ornithine decarboxylase 1 (ODC1) and determination of testosterone metabolizing and phase II conjugating enzymes in the liver. After submission of all study reports to OECD by participants uncoding revealed the following results: (A) When assessing androgenic potential in castrated rats, administration of TREN increased the weights of ventral prostate (VP), seminal vesicles (SV), glans penis, levator ani and bulbocavernosus muscles, and Cowper's glands at the high dose. A similar or stronger (VP, SV) increase of ASTWs was observed for TP; NP and DNP were ineffective. TREN dose-dependently increased gene expression of ODC1 and PBPC3, TP induced expression of these genes even more strongly (almost) to the level of untreated intact animals, whereas NP and DNP were inactive. Liver enzyme activities depending on physiological androgen levels were lower in castrated than in intact rats and could not be restored by androgen treatment. (B) When assessing antiandrogenic potential in TP-supplemented castrated rats, administration of LIN and p,p'-DDE decreased ASTWs only at the high dose. FLUT even more effectively decreased ASTWs, NP and DNP were again without effect. Decreases in androgen-responsive gene expression in the prostate corresponding to the organ weight changes were only observed for p,p'-DDE (high dose) and flutamide (PBPC3 only). p,p'-DDE dose-dependently induced liver weights and most liver enzyme activities including androgen-dependent ones. Our study accurately reproduced ASTW changes obtained in previous studies also under code suggesting that the Hershberger assay is a robust tool to screen for an (anti)androgenic potential. Assessment of ODC1 and PBPC3 gene expression in prostate, however, may only represent a sensitive tool for the detection of an androgenic potential. Finally, p,p'-DDE may affect ASTWs by several mechanisms including enhanced testosterone metabolism.

OECD validation of the Hershberger assay in Japan: Phase 3. Blind study using coded chemicals.

The Organization for Economic Co-operation and Development (OECD) has initiated the development of new guidelines for the screening and testing of potential endocrine disrupters. The Hershberger assay is one of the assays selected for validation based on the need for in vivo screening to detect androgen agonists or antagonists by measuring the response of five sex accessory organs and tissues of castrated juvenile male rats: the ventral prostate, the seminal vesicles with coagulating glands, the levator ani and bulbocavernosus muscle complex (LABC), Cowper's glands, and the glans penis. The Phase 1 feasibility demonstration stage of the Hershberger validation program has been successfully completed with a single androgen agonist and a single antagonist as reference substances. The Phase 2 validation study was performed, employing a range of additional androgen agonists and antagonists. Recently, the Phase 3 validation study was conducted and performed in several International laboratories. Three Japanese laboratories have contributed to the blind study using coded materials of Phase 3 validation. Four coded test substances in the agonistic version and seven substances in the antagonistic version were orally administered by gavage for 10 consecutive days, respectively. In the antagonist version of the assay, 0.2mg/kg/day of testosterone propionate (TP) was coadministered by subcutaneous injection. All five accessory sex reproductive organs and tissues consistently responded with statistically significant changes in weight within a narrow window in both versions. Therefore, the Japanese studies support the Hershberger assay as a reliable and reproducible screening assay for the detection of androgen agonistic and antagonistic effects.

Evaluation of a rapid in vitro androgen receptor transcriptional activation assay using AR-EcoScreen cells.

An accurate and reliable in vitro assay system has been needed for first tier screening of endocrine disrupting chemicals. For the purpose, we previously developed stable AR-EcoScreen cell lines to assess androgen receptor (AR)-mediated transcriptional activation. In this report, we evaluated AR-EcoScreen cell lines as the phase I of prevalidation study by determining the intra-laboratory reproducibility, assay stability, and overall protocol performance of AR-EcoScreen assays. Forty compounds recommended by the ICCVAM were tested for AR agonist and antagonist activity. The mean coefficient of variation (CV) for intra-assay reproducibility in the AR agonist assay was 4.35% for 5alpha-dihydrotestosterone (DHT), and that for the antagonist assay was 5.51% for hydroxyflutamide. The detection limit of the agonist assay was 2.3x10(-11) M for 5alpha-dihydrotestosterone. Furthermore, we examined the overall performance of the method by comparing the predicted result with the ICCVAM classification. Thus, the overall sensitivity, specificity, and accuracy of the agonist assay were 89%, 94%, and 91%, respectively. For the antagonist assay, these values were 94%, 100%, and 96%, respectively. In summary, we concluded that AR-EcoScreen method was ready to proceed to the phase II prevalidation study to asses the inter-laboratory variability and transfer of the protocol.

Development of a high-performance reporter plasmid for detection of chemicals with androgenic activity.

A number of chemicals are present in the environment, and some synthetic chemicals may disrupt endocrine function of wild animals and humans. An effective procedure to screen chemicals for endocrine modulating activity has been needed to ensure the safety of chemicals, and the reporter gene assay technique may provide a powerful tool for screening endocrine-disrupting chemicals. We have developed a high-performance reporter plasmid that can trigger high androgen-dependent induction with high selectivity by using mouse mammary tumor virus (MMTV) androgen-responsive elements and a partial fragment of the rat alpha(2u)-globulin promoter region. This new type plasmid can induce higher transcriptional activation than a commercial PGV-P-based construct bearing the SV40 promoter fragment, and the basal induction level of this plasmid is much lower than that of the PGV-P-based construct. Moreover, only androgen derivatives could selectively induce a high response in the reporter gene assay with the new reporter plasmid. This new type of reporter plasmid, ARE-AUG- Luc+, should be of value in endocrine research and in screening to identify endocrine-modulating chemicals.

[Hot flashes and hormonal treatment of prostate cancer]. / Bouffées de chaleur et traitement hormonal des cancers de la prostate.

Androgen suppression in the context of the treatment of prostatic cancer is responsible for hot flashes in 75% of patients, which alter the quality of life to varying degrees depending on the patient. They constitute a source of major discomfort in 30 to 40% of patients. The pathophysiology of this effect is now known and involves: sex steroids, central opiates and intrahypothalamic catecholamines. The incidence of hot flashes appears to vary according to the type of hormonal treatment administered. The various treatments available are not equally effective. Non-hormonal treatments are of little value. Hormonal treatments: oestrogens and steroidal antiandrogens are the most effective. Progestogens also appear to be just as effective or even more effective than these other agents, with negligible adverse effects at the doses used in this indication.

Antiandrogenic steroidal sulfonyl heterocycles. Utility of electrostatic complementarity in defining bioisosteric sulfonyl heterocycles.

Complementarity of electrostatic potential surface maps was utilized in defining bioisosteric steroidal androgen receptor antagonists. Semiempirical and ab initio level calculations performed on a series of methanesulfonyl heterocycles indicated the requirement for a partial negative charge at the heteroatom attached to C-3 of the steroid nucleus to attain androgen receptor affinity. Synthesis and testing of six heterocycle A-ring-fused dihydroethisterone derivatives support this hypothesis, and we have identified two new androgen receptor antagonists of this class.

[Clinical aspects of the anti-androgens]. / Kliniese aspekte van die anti-androgene.

The ongoing development and gradual availability of the new anti-androgens hold exciting clinical implications for the future. The biosynthesis and interchangeability of the sex steroids, the roles of the ovaries and adrenals and the value and interpretation of biochemical assays in clinical practice are briefly discussed. Because the anti-androgens are used primarily for seborrhoea/acne/hirsutism/oligomenorrhoea, the pathophysiological basis of this socially debilitating syndrome is discussed. The classification of the anti-androgens, their indications, side-effects, dosage schemes and results of treatment are reviewed. Finally, a summary of a possible clinical management regimen is presented.