Investigations on the dose-response relationship of combined exposure to low doses of three anti-androgens in Wistar rats.

The current investigation examines whether combined exposure to three anti-androgens (flutamide, prochloraz, vinclozolin) result in interference with endocrine homeostasis when applied at very low dose levels, and whether the results of combined exposure are more pronounced than to the individual compounds. A pre-post-natal in vivo study design was chosen with more parameters than regulatory testing protocols require (additional endpoints addressing hormone levels, morphology and histopathological examinations). Dose levels were chosen to represent the lowest observed adverse effect level (LOAEL), the no observed adverse effect level (NOAEL), and the acceptable daily intake for each individual substance. Anti-androgenic changes were observable at the effect level (LOAEL) but not at lower exposures. Nipple/areola counts appeared to be a sensitive measure of effect, in addition to male sex organ weights at sexual maturation, and finally gross findings. The results indicate the absence of evidence for effects at low or very low dose levels. No (adverse) effects were seen at the NOAEL dose. A non-monotonic dose-response relationship was not evident. Combined exposure at LOAEL level resulted in enhanced responses for anogenital index, number of areolas/nipples, delayed preputial separation and reduced ventral prostate weight in comparison to the individual compounds.

Parallel assessment of the effects of bisphenol A and several of its analogs on the adult human testis.

STUDY QUESTION: Are bisphenol A (BPA) and BPA analogs (BPA-A) safe for male human reproductive function? SUMMARY ANSWER: The endocrine function of human testes explants [assessed by measuring testosterone and insulin-like factor 3 (INSL3)] was impacted by exposure of the human adult testis explants to BPA/BPA-A. WHAT IS KNOWN ALREADY: The few epidemiologic studies performed suggest that bisphenols have potential endocrine disruptive properties, but they did not identify clear and direct patterns of endocrine disruption. STUDY DESIGN, SIZE, DURATION: Adult human testis explants in culture were exposed to BPA and the analogs bisphenol F (BPF), bisphenol S (BPS), bisphenol E (BPE), bisphenol B (BPB) and bisphenol A diglycidyl ether (BADGE) at 10-9-10-5 M for 24 or 48 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human adult testes were obtained from prostate cancer patients who had no hormone therapy, or from multiorgan donors. After ex vivo exposure to the investigated bisphenols, the measured outcomes were related to histopathology (gross morphology and germ cell viability determined by anti-caspase three immunohistochemistry), and the levels of testosterone, INSL3 and inhibin B were measured using immunoassays. The levels of mRNA encoding key enzymes of bisphenol biotransformation were investigated by quantitative PCR: UGT2B15 UDP (glucuronosyltransferase two family, polypeptide B15), GUSB (glucuronidase beta), SULT1A1 and 3 (sulfotransferase family 1 A member 1 and 3) and STS (steroid sulfatase). MAIN RESULTS AND THE ROLE OF CHANCE: A significant dose-dependent inhibition was found between testosterone levels measured in the culture medium and concentrations of BPA (P = 0.00778 at 24 h and P = 0.0291 at 48 h), BPE (P = 0.039) and BPF (P = 0.00663). The observed BPA and BPA-A-induced inhibition of testosterone production varied according to duration of exposure and BPA/BPA-A concentrations. BPA (10-9 M; P < 0.05), BPB (10-9 M; P < 0.05), BPS (10-9 and 10-8 M; P < 0.05) and BADGE (10-5 M; P < 0.05) increased Leydig cell INSL3 production. By contrast, BPE dose dependently inhibited INSL3 (P = 0.0372). Conversely, Sertoli cell function (inhibin B) and germ cell viability were not significantly affected by either bisphenols. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Environmental compounds cannot be deliberately administered to men, justifying the use of an ex vivo approach. A relatively low number of testes samples were available for analysis (n = 3, except for testosterone secretion with n = 5). The active concentrations of BPA and BPA-A used in the study were higher than those found in human biological fluids. WIDER IMPLICATIONS OF THE FINDINGS: Under our experimental conditions, direct exposure to BPA or BPA-A can result in endocrine disturbance in the adult human testis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Inserm (Institut National de la Santé et de la Recherche Médicale), EHESP-School of Public Health, University of Rennes1, by grants from the Agence Nationale de la Recherche (ANR; grant#ANR-13-CESA-0012-03 NEWPLAST) and Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail (ANSES; grant#EST-2010/2/046 (BPATESTIS)). All authors declare they have no current or potential competing financial interests.

Biological responses to phenylurea herbicides in fish and amphibians: New directions for characterizing mechanisms of toxicity.

Urea-based herbicides are applied in agriculture to control broadleaf and grassy weeds, acting to either inhibit photosynthesis at photosystem II (phenylureas) or to inhibit acetolactate synthase acetohydroxyacid synthase (sulfonylureas). While there are different chemical formulas for urea-based herbicides, the phenylureas are a widely used class in North America and have been detected in aquatic environments due to agricultural run-off. Here, we summarize the current state of the literature, synthesizing data on phenylureas and their biological effects in two non-target animals, fish and amphibians, with a primary focus on diuron and linuron. In fish, although the acutely lethal effects of diuron in early life stages appear to be >1mg/L, recent studies measuring sub-lethal behavioural and developmental endpoints suggest that diuron causes adverse effects at lower concentrations (i.e. <0.1mg/L). Considerably less toxicity data exist for amphibians, and this is a knowledge gap in the literature. In terms of sub-lethal effects and mode of action (MOA), linuron is well documented to have anti-androgenic effects in vertebrates, including fish. However, there are other MOAs that are not adequately assessed in toxicology studies. In order to identify additional potential MOAs, we conducted in silico analyses for linuron and diuron that were based upon transcriptome studies and chemical structure-function relationships (i.e. ToxCast™, Prediction of Activity Spectra of Substances). Based upon these analyses, we suggest that steroid biosynthesis, cholesterol metabolism and pregnane X receptor activation are common targets, and offer some new endpoints for future investigations of phenylurea herbicides in non-target animals.

Comparing effect levels of regulatory studies with endpoints derived in targeted anti-androgenic studies: example prochloraz.

Prochloraz is an imidazole fungicide, and its regulatory toxicological data package has been primarily generated in the 1990s. More recently, studies have been published demonstrating an interaction with hormone receptors/steroidogenesis and effects with an endocrine mode of action. In the present study, prochloraz has been investigated in a comprehensive in vivo study including relevant elements of current regulatory reproduction toxicity studies and additional mechanistic parameters. Prochloraz was administered per gavage in oil from GD 6 to PND 83 to pregnant and lactating Wistar rats and their respective offspring, at doses of 0.01 mg/kg bw/day (acceptable daily intake of prochloraz), 5 mg/kg bw/day [expected no-observed-effect-level (NOEL)] and 30 mg/kg bw/day. At 30 mg/kg bw/day maternal and offspring effects (decreased viability, lower number of live offspring) were seen including a delayed entry into male puberty (+1 day) accompanied by lower male offspring body weights, increased anogenital distance/index in females and transiently retained nipples in males at PND 12 (not seen at PND 20). The only finding at the "expected NOEL" was increased incidences of transiently retained nipples in males which are not considered adverse. No effects were seen in the low-dose group. There was no evidence for a non-monotonic dose-response curve or effects at low levels.

Disruption of rat testis development following combined in utero exposure to the phytoestrogen genistein and antiandrogenic plasticizer di-(2-ethylhexyl) phthalate.

Fetal exposure to environmental endocrine disruptors (EDs) is thought to contribute to reported idiopathic increases in adult male reproductive abnormalities. Although humans are exposed to myriad EDs from conception to adulthood, few studies have evaluated the effects of combined EDs on male reproduction. In the present study, we demonstrate that simultaneous gestational exposure to the phytoestrogen genistein and the antiandrogenic plasticizer di-(2-ethyhexyl) phthalate (DEHP) induces long-term alterations in testis development and function. Pregnant Sprague Dawley rats were gavaged from Gestational Day 14 to birth with corn oil, genistein, DEHP, or their mixture at 10 mg/kg/day, a dose selected from previous dose-response studies using single chemicals for its lack of long-term testicular effects. Hormonal and testicular end points were examined in adult male offspring. Serum testosterone levels were unchanged. However, significant increases were observed in testis weight and in the expression of mast cell markers in testes from adult rats exposed gestationally to combined compounds. The ED mixture also altered the mRNA expression of Sertoli cell makers Wt1 and Amh and germ cell markers cKit and Sox17, measured by quantitative real-time PCR (qPCR), suggesting long-term disruption in testis function and spermatogenesis. Alterations in germ cell markers might reflect direct effects on fetal gonocytes or indirect effects via primary targeting of somatic cells, as suggested by differentially regulated Leydig cell associated genes (Hsd3b, Anxa1, Foxa3, and Pdgfra), determined by gene expression array, qPCR, and protein analyses. The two chemicals, when given in combination, induced long-term reproductive toxicity at doses not previously reported to produce any conspicuous long-term effects. Our study therefore highlights a need for a more comprehensive evaluation of the effects of ED mixtures.

Mixture effects at very low doses with combinations of anti-androgenic pesticides, antioxidants, industrial pollutant and chemicals used in personal care products.

Many xenobiotics have been identified as in vitro androgen receptor (AR) antagonists, but information about their ability to produce combined effects at low concentrations is missing. Such data can reveal whether joint effects at the receptor are induced at low levels and may support the prioritisation of in vivo evaluations and provide orientations for the grouping of anti-androgens in cumulative risk assessment. Combinations of 30 AR antagonists from a wide range of sources and exposure routes (pesticides, antioxidants, parabens, UV-filters, synthetic musks, bisphenol-A, benzo(a)pyrene, perfluorooctane sulfonate and pentabromodiphenyl ether) were tested using a reporter gene assay (MDA-kb2). Chemicals were combined at three mixture ratios, equivalent to single components' effect concentrations that inhibit the action of dihydrotesterone by 1%, 10% or 20%. Concentration addition (CA) and independent action were used to calculate additivity expectations. We observed complete suppression of dihydrotestosterone effects when chemicals were combined at individual concentrations eliciting 1%, 10% or 20% AR antagonistic effect. Due to the large number of mixture components, the combined AR antagonistic effects occurred at very low concentrations of individual mixture components. CA slightly underestimated the combined effects at all mixture ratios. In conclusion, large numbers of AR antagonists from a wide variety of sources and exposure routes have the ability of acting together at the receptor to produce joint effects at very low concentrations. Significant mixture effects are observed when chemicals are combined at concentrations that individually do not induce observable AR antagonistic effects. Cumulative risk assessment for AR antagonists should apply grouping criteria based on effects where data are available, rather than on criteria of chemical similarity.