Erika Rosivatz

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.

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.

Removal or masking of phosphatidylinositol(4,5)bisphosphate from the outer mitochondrial membrane causes mitochondrial fragmentation

Mitochondria are central players in programmed cell death and autophagy. While phosphoinositides are well established regulators of membrane traffic, cellular signalling and the destiny of certain organelles, their presence and role for mitochondria remain elusive. In this study we show that removal of PtdIns(4,5)P2 by phosphatases or masking the lipid with PH domains leads to fission of mitochondria and increased autophagy. Induction of general autophagy by amino acid starvation also coincides with the loss of mitochondrial PtdIns(4,5)P2, suggesting an important role for this lipid in the processes that govern mitophagy. Our findings reveal that PKCα can rescue the removal or masking of PtdIns(4,5)P2, indicating that the inositol lipid is upstream of PKC.

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

Identification of Cyclin A2 as the Downstream Effector of the Nuclear Phosphatidylinositol 4,5-Bisphosphate Signaling Network

In addition to the well characterized phosphoinositide second messengers derived from the plasma membrane, increasing evidence supports the existence of a nuclear phosphoinositide signaling network. The aim of this investigation was to dissect the role played by nuclear phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in cell cycle progression and to determine the cell cycle regulatory component(s) that are involved. A number of cytosolic/nuclear PtdIns(4,5)P2-deficient Swiss 3T3 cell lines were established, and their G0/G1/S cell cycle phase transitions induced by defined mitogens were examined. Our results demonstrate that nuclear PtdIns(4,5)P2 down-regulation caused a delay in phorbol ester-induced S phase entry and that this was at least in part channeled through cyclin A2 at the transcriptional level. In summary, these data identify cyclin A2 as a downstream effector of the nuclear PtdIns(4,5)P2 signaling network and highlight the importance of nuclear PtdIns(4,5)P2 in the regulation of mammalian mitogenesis.

The novel molecule 2-(5-(2-chloroethyl)-2-acetoxy-benzyl)- 4-(2-chloroethyl)-phenyl acetate inhibits phosphoinositide 3-kinase⁄Akt⁄mammalian target of rapamycin signalling through JNK activation in cancer cells

Screening a compound library of compound 48/80 analogues, we identified 2‐[5‐(2‐chloroethyl)‐2‐acetoxy‐benzyl]‐4‐(2‐chloroethyl)‐phenyl acetate (E1) as a novel inhibitor of the phosphoinositide 3‐kinase/Akt pathway. In order to determine the mechanism of action of E1, we analysed the effect of E1 on components of the phosphoinositide 3‐kinase/Akt/mammalian target of rapamycin (mTOR) pathway. E1 demonstrated dose‐dependent and time‐dependent repression of Akt and mTOR activity in prostate and breast cancer cell lines, PC‐3 and MCF‐7, respectively. Inhibition of Akt and mTOR activity by E1 also coincided with increased c‐Jun NH2‐terminal kinase (JNK) phosphorylation. However, the mode of action of E1 is different from that of the mTOR inhibitor rapamycin. Proliferation and cell cycle analysis revealed that E1 induced cell cycle arrest and cell death in PC‐3 and MCF‐7 cells. Moreover, pretreatment of cancer cells with the JNK inhibitor SP600125 abolished the repression of Akt and mTOR activity by E1, indicating that the inhibition of Akt and mTOR by E1 is mediated through JNK activation. Consistently, E1 repressed Akt and mTOR activity in wild‐type and p38‐null mouse embryonic fibroblasts (MEFs), but not in MEFs lacking JNK1/2, and JNK‐null MEFs were less sensitive to the antiproliferative effects of E1. We further showed that E1 can function cooperatively with suboptimal concentrations of paclitaxel to induce cell death in PC‐3 and MCF‐7 cells. Taken together, these data suggest that E1 induces cancer cell death through the JNK‐dependent repression of Akt and mTOR activity and may provide a valuable compound for further development and research.

Imaging the boundaries—innovative tools for microscopy of living cells and real-time imaging

Recently, light microscopy moved back into the spotlight, which is mainly due to the development of revolutionary technologies for imaging real-time events in living cells. It is truly fascinating to see enzymes “at work” and optically acquired images certainly help us to understand biological processes better than any abstract measurements. This review aims to point out elegant examples of recent cell-biological imaging applications that have been developed with a chemical approach. The discussed technologies include nanoscale fluorescence microscopy, imaging of model membranes, automated high-throughput microscopy control and analysis, and fluorescent probes with a special focus on visualizing enzyme activity, free radicals, and protein–protein interaction designed for use in living cells.

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

Spatial Localization of PtdIns P 2 in Phase-Separated Giant Unilamellar Vesicles with a Fluorescent PLC-delta 1 PH Domain

This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate that are larger than 20 microm in size. The phospholipids composition of the vesicular membrane is such that fluid lamellar and liquid-ordered or gel phases are formed and separate within the confines of one vesicle. It outlines the preparation of a protein fluorescent label, pleckstrin homology domain from phospholipase C-delta 1, that binds specifically to phosphatidylinositol 4,5-bisphosphate. Using fluorescence microscopy, the presence and spatial position of this phosphorylated phosphatidylinositol lipid on the lipid membrane have been located with the pleckstrin homology domain. We show that phosphatidylinositol 4,5-bisphosphate and the phospholipase C-delta 1 pleckstrin homology domain are located to the fluid phase of the vesicle membrane. This approach can therefore show how membrane physical properties can affect enzyme binding to phosphatidylinositol 4,5-bisphosphate and thus further the understanding of important membrane processes such as endocytosis.

Measurement of PTEN activity in vivo by imaging phosphorylated Akt.

This chapter describes an indirect approach to measure PTENs lipid phosphatase activity in vivo. PTEN counteracts phosphatidylinositol 3-kinase action in dephosphorylating 3-phosphorylated phosphoinositides. Therefore, PtdIns(3,4,5)P3-dependent activation and phosphorylation of the survival kinase Akt can be used as readout for cellular PTEN activity. Here we have outlined a detailed procedure employing a phosphoserine-specific anti-Akt antibody to examine the content of phosphorylated Akt by immunofluorescence and its dependence on PTEN activity.