Frances Orton

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.

Effects of nitrate and atrazine on larval development and sexual differentiation in the northern leopard frog Rana pipiens

Pollution from agrochemicals may be contributing to the global decline in amphibian populations. Environmentally relevant concentrations of nitrate and/or atrazine on anuran development and gonadal differentiation were tested. Four replicates of 20 tadpoles per tank (80/treatment) were exposed from Taylor-Kollros stage 2 to 3 to stage 23 to 34 to either 10 mg/L nitrate, 10 microg/L atrazine, a combined exposure of 10 mg/L nitrate plus 10 microg/L atrazine, or untreated controls. No treatment-dependent effects on weight, snout-vent or hind limb length, or time to forelimb emergence were observed. The proportions of females increased in all treatments compared to the controls, especially in the combined treatment (chi2 = 17.90, df = 6, p = 0.0065, combined = 66.4% female, control = 41% female). The frequency of intersex was low in all treatments. No treatment-related effects on the total number of spermatogenic cells were observed, but the ratio of cell types differed in that testes from animals in the treated groups exhibited more spermatogonia, fewer spermatocytes, and more spermatids than the control (significantly different, Kruskal-Wallis, p < 0.05). Ovaries from animals treated with nitrate or atrazine exhibited larger immature (previtellogenic) and mature (vitellogenic) follicles, but ovaries from the combined treatment had larger immature follicles only. Testicular oocytes were observed in the nitrate-only and atrazine-only treatments, and the control treatment, but not the combined treatment. Overall, this study has demonstrated changes in sex ratios that are more marked in response to combined nitrate/atrazine exposure than with these chemicals alone. Histological evidence suggests that premature maturation of gonad may occur as a result of nitrate and/or atrazine exposure during larval development.

Batrachochytrium dendrobatidis infection and lethal chytridiomycosis in caecilian amphibians (Gymnophiona)

Batrachochytrium dendrobatidis (Bd) is commonly termed the ‘amphibian chytrid fungus’ but thus far has been documented to be a pathogen of only batrachian amphibians (anurans and caudatans). It is not proven to infect the limbless, generally poorly known, and mostly soil-dwelling caecilians (Gymnophiona). We conducted the largest qPCR survey of Bd in caecilians to date, for more than 200 field-swabbed specimens from five countries in Africa and South America, representing nearly 20 species, 12 genera, and 8 families. Positive results were recovered for 58 specimens from Tanzania and Cameroon (4 families, 6 genera, 6+ species). Quantities of Bd were not exceptionally high, with genomic equivalent (GE) values of 0.052–17.339. In addition, we report the first evidence of lethal chytridiomycosis in caecilians. Mortality in captive (wild-caught, commercial pet trade) Geotrypetes seraphini was associated with GE scores similar to those we detected for field-swabbed, wild animals.

Do hormone-modulating chemicals impact on reproduction and development of wild amphibians?

Globally, amphibians are undergoing a precipitous decline. At the last estimate in 2004, 32% of the approximately 6000 species were threatened with extinction and 43% were experiencing significant declines. These declines have been linked with a wide range of environmental pressures from habitat loss to climate change, disease and pollution. This review evaluates the evidence that endocrine‐disrupting contaminants (EDCs) – pollutants that affect hormone systems – are impacting on wild amphibians and contributing to population declines. The review is limited to anurans (frogs and toads) as data for effects of EDCs on wild urodeles (salamanders, newts) or caecilians (limbless amphibians) are extremely limited. Evidence from laboratory studies has shown that a wide range of chemicals have the ability to alter hormone systems and affect reproductive development and function in anurans, but for the most part only at concentrations exceeding those normally found in natural environments. Exceptions can be found for exposures to the herbicide atrazine and polychlorinated biphenyls in leopard frogs (Rana pipiens) and perchlorate in African clawed frogs (Xenopus laevis). These contaminants induce feminising effects on the male gonads (including ‘intersex’ – oocytes within testes) at concentrations measured in some aquatic environments. The most extensive data for effects of an EDC in wild amphibian populations are for feminising effects of atrazine on male gonad development in regions across the USA. Even where strong evidence has been provided for feminising effects of EDCs, however, the possible impact of these effects on fertility and breeding outcome has not been established, making inference for effects on populations difficult. Laboratory studies have shown that various chemicals, including perchlorate, polychlorinated biphenyls and bromodiphenylethers, also act as endocrine disrupters through interfering with thyroid‐dependent processes that are fundamental for amphibian metamorphosis. Perchlorate has also been shown to induce these effects in wild anuran populations from perchlorate‐contaminated environments. Overall, the published data available suggest that some health effects observed in wild anuran populations, most notably intersex, likely have a chemical aetiology; however they derive only from very few anuran species and for a few pesticides at field sites in the USA. To understand better the impacts of EDCs on wild anuran populations, as well as other amphibian groups, assessment of fertility in exposed animals are required. Development of non‐destructive biomarkers that are indicative of specific EDC‐effect mechanisms are also needed to allow the study of vulnerable populations. This will help to distinguish the effects of EDCs from other environmental and/or genetic influences on development and reproduction.

Agricultural intensity in ovo affects growth, metamorphic development and sexual differentiation in the Common toad (Bufo bufo)

Pollution was cited by the Global Amphibian Assessment to be the second most important cause of amphibian decline worldwide, however, the effects of the agricultural environment on amphibians are not well understood. In this study, spawn from Bufo bufo was taken from four sites in England and Wales with varying intensities of arable agriculture. Spawn was either placed in tanks containing aged tap water (ex-situ, five replicates) or in cages at the native site (caged, five replicates). Hatching success, abnormal tadpoles, and forelimb emergence were recorded during the larval stage. Individuals were also sampled at five time points (TP) during development (5-, 7-, 9-, 12-, 15-weeks post-hatch) and analysed for morphological parameters. The thyroids (TP2) and the gonads (TP3,4,5) were also analysed histologically. With the exception of the thyroid histopathology, all analysed endpoints were significantly different between ex-situ individuals reared under identical conditions from the different sites. In addition, intensity of arable agriculture had a negative effect on growth and development. At one site, despite distinct rearing conditions, a high level of intersex (up to 42%) and similar sex ratios were observed in both ex-situ and caged individuals. Taken together, these data suggest that maternal exposure and/or events in ovo had a much larger effect on growth, metamorphic development, and sexual differentiation in B. bufo than the ambient environment. This could have important implications for traditional exposure scenarios that typically begin at the larval stage. Intersex is reported for the first time in European amphibians in situ, highlighting the potential use of distinct populations of amphibians in fundamental research into the aetiology of specific developmental effects in wild amphibians.

Competitive Androgen Receptor Antagonism as a Factor Determining the Predictability of Cumulative Antiandrogenic Effects of Widely Used Pesticides

Background: Many pesticides in current use have recently been revealed as in vitro androgen receptor (AR) antagonists, but information about their combined effects is lacking. Objective: We investigated the combined effects and the competitive AR antagonism of pesticide mixtures. Methods: We used the MDA-kb2 assay to test a combination of eight AR antagonists that did not also possess AR agonist properties (“pure” antagonists; 8 mix: fludioxonil, fenhexamid, ortho-phenylphenol, imazalil, tebuconazole, dimethomorph, methiocarb, pirimiphos-methyl), a combination of five AR antagonists that also showed agonist activity (5 mix: cyprodinil, pyrimethanil, vinclozolin, chlorpropham, linuron), and all pesticides combined (13 mix). We used concentration addition (CA) and independent action (IA) to formulate additivity expectations, and Schild plot analyses to investigate competitive AR antagonism. Results: A good agreement between the effects of the mixture of eight “pure” AR antagonists and the responses predicted by CA was observed. Schild plot analysis revealed that the 8 mix acted by competitive AR antagonism. However, the observed responses of the 5 mix and the 13 mix fell within the “prediction window” boundaries defined by the predicted regression curves of CA and IA. Schild plot analysis with these mixtures yielded anomalous responses incompatible with competitive receptor antagonism. Conclusions: A mixture of widely used pesticides can, in a predictable manner, produce combined AR antagonist effects that exceed the responses elicited by the most potent component alone. Inasmuch as large populations are regularly exposed to mixtures of antiandrogenic pesticides, our results underline the need for considering combination effects for these substances in regulatory practice.

Response to A critique of the European Commission Document, "State of the Art Assessment of Endocrine Disrupters" by Rhomberg and colleagues--letter to the editor.

The European Commission is in the process of preparing regulatory activities for endocrine disrupting chemicals. In support of this process, the European Commission has asked for a summary of the state of endocrine disrupter science which was to be completed within 12 months. A draft version of our science summary was published by the European Commission in the summer of 2011, with the aim of generating feedback from stakeholders. We have received comments from EU member state authorities and from the European Chemical Industry (CEFIC, ECPA) and these were all taken into account in the final version of the science summary. The final version of the science summary was submitted as an annex to our final SOTA ED (Kortenkamp et al., 2011) which was completed on 23 December 2011. In their American Chemistry Council commissioned critique, Rhomberg et al. (2012) focus entirely on the draft version of the science summary, but largely ignore the main body of our report. Rhomberg et al. argue that because we did not use methodological approaches needed for comprehensive substance assessments, our report must be biased. This line of argumentation is deeply flawed. It might be the result of a lack of understanding of the distribution of competences in the European Union which the authors (all of them US or Canadian nationals) may not be familiar with. Rhomberg et al. seem to have assumed that the European Commission would base their policy initiatives on detailed risk assessments of a large number of chemicals, in terms of their endocrine disrupting properties. But the European Commission (the executive arm of the European Union) itself is not responsible for risk assessments of chemicals. Rather, it shapes the general principles of European Union chemicals policy of which risk assessment is but one element. Accordingly, the European Commission did not ask for detailed and in-depth risk assessments for a large number of chemicals to support their policy initiatives, nor is such an analysis needed as a foundation for policy. In the European Union, the task of detailed chemical evaluations and risk assessments falls in the first instance to industry and under well defined circumstances to European agencies such as the European Food Safety Authority or the European Chemicals Agency and to competent authorities of European Union member states. What the European Commission needed, and what it requested us to prepare, was a summary of endocrine disrupter science, primarily with the aim of assessing whether policy initiatives in this area are scientifically justified and called for. Accordingly, they commissioned an assessment of whether endocrine disruption is a problem, and whether there are indications that chemical exposures play a role in endocrine-related health outcomes or wildlife effects. A specific objective in relation to a clause of the European chemicals regulation REACH was to evaluate the scientific basis for the notion that endocrine disrupting substances might cause effects of a concern equivalent to the hazards posed by carcinogens, mutagens and reproductive and developmental toxicants. In line with these requirements, we prepared a science summary to assess the plausibility that xenobiotics might play a role in the aetiology of various health endpoints potentially related to endocrine disrupters. To achieve this aim, our assessment of the scientific evidence was based on the principles proposed by WHO/IPCS (2002) as a basis for attribution of effects to endocrine disruption (report chapter 3.16, p32). In dealing with this problem, it would have been inappropriate to utilize the causal criteria for assessing endocrine disrupters described in chapter 7 of the same report. This seems to have been entirely misunderstood by Rhomberg and colleagues. Rhomberg et al. have also overlooked the fact that we have dealt extensively with the issue of weight of evidence, both in the science summary and in the main part of our report (Kortenkamp et al., 2011). They ignore the complexity of developing weight of evidence approaches and seem to assume that such approaches are already available and agreed upon. However, this is not the case. As we have stressed in our report (Kortenkamp et al., 2011), weight of evidence approaches for endocrine disrupters are yet to Letter to the editor

A novel biomarker for anti-androgenic activity in placenta reveals risks of urogenital malformations

It has been hypothesized that the rise in male reproductive disorders over recent decades may at least be partially attributable to environmental factors, including chemical exposures, but observed associations with single chemicals were rather weak. The aim of this case-control study was to explore the relationship between exposure to mixtures of (anti-)androgenic chemicals during pregnancy and the risk of cryptorchidism and/or hypospadias in offspring, using the total effective xenobiotic burden of anti-androgens (TEXB-AA) as a biomarker. A subsample of 29 cases (16 of cryptorchidism, 12 of hypospadias, and one of both disorders) and 60 healthy controls was nested in a cohort of male newborns recruited between October 2000 and July 2002. The (anti-)androgenic activity of placenta samples collected at delivery was assessed using TEXB-AA biomarker, combined with a bioassay-directed fractionation protocol that separated endogenous hormones from most (anti-)androgenic chemicals by normal-phase HPLC. The bioassay measures the androgen-induced luciferase activity and the inhibition of this pathway by (anti-)androgens. First, we collected 27 HPLC fractions in each placenta extract, which were all tested in the bioassay. The multivariable statistical analyses indicated a statistically significant positive dose-response association between the potent anti-androgenic activity of the HPLC fraction collected during minutes 1-2 (F2) and the risk of malformations (odds ratio: 2.33, 95% CI: 1.04-5.23). This study represents a novel approach for the estimation of combined effects of the total anti-androgenic load and the associations suggest an effect of environmental pollutants on the development of fetal reproductive tract.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/149/6/605/suppl/DC1.

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