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.

Metabolic and reproductive effects of relatively low concentrations of beclomethasone dipropionate, a synthetic glucocorticoid, on fathead minnows.

Pharmaceuticals present in the aquatic environment could adversely affect aquatic organisms. Synthetic glucocorticoids (GC) are used in large quantities as anti-inflammatory drugs and have been reported to be present in river water. In order to assess the impact of environmental concentrations of GCs, an in vivo experiment was conducted with adult fathead minnows. Fish were exposed to 0.1 µg/L, 1 µg/L, or 10 µg/L beclomethasone dipropionate (BCMD) via a flow-through system over a period of 21 days. Similar duplicate tanks served as control, with no chemical added. There was a concentration-related increase in plasma glucose concentration and a decrease in blood lymphocyte count. Induction of male secondary sexual characters and a decreasing trend in plasma vitellogenin (Vtg) concentrations in female fish were observed with increasing exposure concentration of BCMD. Expression profiles of selected genes (phosphoenolpyruvate carboxykinase - PEPCK, glucocorticoid receptor - GR, and Vtg) in liver also demonstrated concentration-related effects at all three tested concentrations. The results suggest that GCs could cause effects in lower micrograms per liter concentrations that could be environmentally relevant for total GCs present in the environment. Therefore, studies to determine the environmental concentrations of GCs and no effect concentrations are needed to assess if GCs pose a risk to the aquatic environment.

Synthetic glucocorticoids in the environment: first results on their potential impacts on fish.

Human pharmaceuticals have been shown to be entering the aquatic environment in quantities that may produce adverse effects to aquatic organisms. This paper investigates the impacts of synthetic glucocorticoids (GCs), which are used in large amounts as anti-inflammatory drugs, on fish. Mammalian cell lines were transiently transfected with trout corticosteroid receptors (GR1, GR2, and MR) and the transactivation abilities of ten of the most prescribed GCs in the UK were measured in vitro. They showed significantly higher activity with GR2 than with GR1. In order to assess any impacts in vivo, adult fathead minnows were exposed to either 1 µg prednisolone/L or 1 µg beclomethasone dipropionate/L for 21 days. Plasma glucose concentrations were increased and leucocytes were reduced significantly in GC-exposed groups compared to the control group. In another experiment, fish were exposed to three different concentrations of Beclomethasone dipropionate and a dose-dependent increase of plasma glucose was found. The results suggest that low concentrations of synthetic GCs present in water could cause adverse effects on fish. Therefore, quantification of GCs in the aquatic environment and the effects of GCs at environmentally relevant concentrations are required in order to determine if GCs pose a threat to wild fish populations.

The consequences of exposure to mixtures of chemicals: Something from 'nothing' and 'a lot from a little' when fish are exposed to steroid hormones.

Ill-defined, multi-component mixtures of steroidal pharmaceuticals are present in the aquatic environment. Fish are extremely sensitive to some of these steroids. It is important to know how fish respond to these mixtures, and from that knowledge develop methodology that enables accurate prediction of those responses. To provide some of the data required to reach this objective, pairs of fish were first exposed to five different synthetic steroidal pharmaceuticals (one estrogen, EE2; one androgen, trenbolone; one glucocorticoid, beclomethasone dipropionate; and two progestogens, desogestrel and levonorgestrel) and concentration-response data on egg production obtained. Based on those concentration-response relationships, a five component mixture was designed and tested twice. Very similar effects were observed in the two experiments. The mixture inhibited egg production in an additive manner predicted better by the model of Independent Action than that of Concentration Addition. Our data provide a reference case for independent action in an in vivo model. A significant combined effect was observed when each steroidal pharmaceutical in the mixture was present at a concentration which on its own would produce no statistically significant effect (something from 'nothing'). Further, when each component was present in the mixture at a concentration expected to inhibit egg production by between 18% (Beclomethasone diproprionate) and 40% (trenbolone), this mixture almost completely inhibited egg production: a phenomenon we term 'a lot from a little'. The results from this proof-of-principle study suggest that multiple steroids present in the aquatic environment can be analysed for their potential combined environmental risk.

Effects of Common Pesticides on Prostaglandin D2 (PGD2) Inhibition in SC5 Mouse Sertoli Cells, Evidence of Binding at the COX-2 Active Site, and Implications for Endocrine Disruption.

BACKGROUND: There are concerns that diminished prostaglandin action in fetal life could increase the risk of congenital malformations. Many endocrine-disrupting chemicals have been found to suppress prostaglandin synthesis, but to our knowledge, pesticides have never been tested for these effects. OBJECTIVES: We assessed the ability of pesticides that are commonly used in the European Union to suppress prostaglandin D2 (PGD2) synthesis. METHODS: Changes in PGD2 secretion in juvenile mouse Sertoli cells (SC5 cells) were measured using an ELISA. Coincubation with arachidonic acid (AA) was conducted to determine the site of action in the PGD2 synthetic pathway. Molecular modeling studies were performed to assess whether pesticides identified as PGD2-active could serve as ligands of the cyclooxygenase-2 (COX-2) binding pocket. RESULTS: The pesticides boscalid, chlorpropham, cypermethrin, cyprodinil, fenhexamid, fludioxonil, imazalil (enilconazole), imidacloprid, iprodione, linuron, methiocarb, o-phenylphenol, pirimiphos-methyl, pyrimethanil, and tebuconazole suppressed PGD2 production. Strikingly, some of these substances-o-phenylphenol, cypermethrin, cyprodinil, linuron, and imazalil (enilconazole)-showed potencies (IC50) in the range between 175 and 1,500 nM, similar to those of analgesics intended to block COX enzymes. Supplementation with AA failed to reverse this effect, suggesting that the sites of action of these pesticides are COX enzymes. The molecular modeling studies revealed that the COX-2 binding pocket can accommodate most of the pesticides shown to suppress PGD2 synthesis. Some of these pesticides are also capable of antagonizing the androgen receptor. CONCLUSIONS: Chemicals with structural features more varied than previously thought can suppress PGD2 synthesis. Our findings signal a need for in vivo studies to establish the extent of endocrine-disrupting effects that might arise from simultaneous interference with PGD2 signaling and androgen action. CITATION: Kugathas S, Audouze K, Ermler S, Orton F, Rosivatz E, Scholze M, Kortenkamp A. 2016. Effects of common pesticides on prostaglandin D2 (PGD2) inhibition in SC5 mouse Sertoli cells, evidence of binding at the COX-2 active site, and implications for endocrine disruption. Environ Health Perspect 124:452-459; http://dx.doi.org/10.1289/ehp.1409544.

From single chemicals to mixtures--reproductive effects of levonorgestrel and ethinylestradiol on the fathead minnow.

The aquatic environment is polluted with thousands of chemicals. It is currently unclear which of these pose a significant threat to aquatic biota. The typical exposure scenario is now represented by a widespread blanket of contamination composed of myriads of individual pollutants-each typically present at a low concentration. The synthetic steroids, 17α-ethinylestradiol and levonorgestrel, have been widely reported to be present in the aquatic environment in the low ng to sub-ng/l range. They are widely used in contraceptive formulations, both individually and in combination. Our research employed the fathead minnow (Pimephales promelas) 21 day 'pair-breeding' assay to assess reproductive output when pairs of fish were exposed to the single chemicals at low environmentally relevant concentrations, and then to a binary mixture of them. A variety of endpoints were assessed, including egg production, which was inhibited in a concentration-dependent manner by both the individual chemicals and the mixture. Significant, sex specific effects were also seen with both chemicals, at differing levels of biological organisation. Plasma concentrations of EE2 and levonorgestrel were predicted and in the case of levonorgestrel measured, and compared with the human therapeutic plasma concentrations (Read-Across approach) to support the interpretation of the results. A novel quantitative method was developed for the data analysis, which ensured a suitable endpoint for the comparative mixture assessment. This approach compares the reproductive performance from individual pairs of fish during chemical exposure to its pre-treatment performance. The responses from the empirical mixture study were compared to predictions derived from the single substance data. We hypothesised combined responses which were best described by the concept of concentration addition, and found no clear indications against this additivity expectation. However, the effect profiles support the current knowledge that both compounds act in different ways to reduce egg production in fish, and suggest that probably response addition (also called Independent action) is the more appropriate mixture model in this case.

Prediction of environmental concentrations of glucocorticoids: the River Thames, UK, as an example.

Synthetic glucocorticoids (GCs) are consumed in large amounts as anti-inflammatory and immunosuppressive drugs worldwide. Based on what has been learnt from studies of other human pharmaceuticals, they are likely to be present in the aquatic environment. However, to date, information on the environmental concentrations of GCs is very limited. The situation is complicated by the fact that a considerable number of GCs are in everyday use in most developed countries. Hence, obtaining a full picture of GC concentrations in the aquatic environment using the traditional analytical chemistry approach would be time-consuming and expensive. Thus, we took a modelling approach to predict the total environmental concentration of all synthetic GCs (consisting of 28 individual GCs) in the River Thames, as a first step in risk assessment of these drugs. Using reliable data on consumption, the LF2000-WQX model predicts mean concentrations up to 30 ng/L of total GCs in surface water as a best case scenario when the lowest excretion and highest removal rates in sewage treatment works were used, whereas mean concentrations up to 850 ng/L were predicted when the highest excretion and lowest removal rates are considered. We also present the 10th and 90th percentile concentrations (which indicate the likely range of concentrations seen from high flow to low flow conditions in the river) of the highest and lowest consumed GCs, to show the spatial and temporal variations of the concentrations of individual GCs. These data probably provide reliable estimates of the likely range of concentrations of GCs in a typical river impacted by effluent from many sewage treatment plants. Results also identify the hot spots where field studies on fish could be focused. To determine if aquatic organisms face any threat from GCs, laboratory toxicity studies should be conducted using concentrations similar to those reported here.