Systematic review of associations of polychlorinated biphenyl (PCB) exposure with declining semen quality in support of the derivation of reference doses for mixture risk assessments.

BACKGROUND: Mixture risk assessments require reference doses for common health endpoints of all the chemicals to be considered together. In support of a mixture risk assessment for male reproductive health, we conducted a systematic review of the literature on associations between exposures to Polychlorinated Biphenyls (PCBs) and declines in semen quality. PCBs can act as Aryl-hydrocarbon Receptor (AhR)-agonists and Androgen Receptor (AR)-antagonists, both mechanisms which can affect sperm parameters. PCBs and other AR-antagonists can produce additive combination effects. Based on these observations our objective was to systematically gather data from animal and human studies to derive a reference dose for declines in semen quality for individual PCB. METHODS: We systematically reviewed and evaluated the evidence in human epidemiological and experimental animal studies on associations between PCBs and deteriorations in semen quality. Human data and findings from animal studies with PCB mixtures were considered as supporting evidence. Information for individual congeners from animal studies was required for inclusion in mixture risk assessment. Using a robust confidence rating approach, we identified suitable studies to derive reference doses for individual PCB congeners. RESULTS: Evaluation of human epidemiological studies revealed several reports of adverse effects on sperm parameters linked to PCB exposures, although some studies reported improved semen quality. Our review of experimental animal studies found that treatments with PCBs affected semen quality, in most cases adversely. We found robust evidence that PCB-118 and -169 were linked to declines in semen quality. Evidence for adverse effects of PCB-126, -132, -149, and -153 was moderate, whereas for PCB-77 it was slight and for PCB-180 indeterminate. Using widely accepted risk assessment procedures, we estimated reference dose values of 0.0029 µg/kg/day for PCB-118 and 0.00533 µg/kg/day for PCB-169. In addition, we derived values for PCB-126: 0.000073 µg/kg/day, PCB-132: 0.0228 µg/kg/day, PCB-149: 0.656 µg/kg/day, and PCB-153: 0.0058 µg/kg/day. CONCLUSIONS: We found robust evidence for links between PCB exposure and deteriorations in semen quality, and derived reference doses for a set of congeners. We intend to use these values in combination with congener-specific exposure data in a mixture risk assessment for declines in semen quality, involving several other antiandrogenic chemicals.

Combined exposures to bisphenols, polychlorinated dioxins, paracetamol, and phthalates as drivers of deteriorating semen quality.

BACKGROUND: Semen quality in men continues to decline in Western countries, but the contours of the issue remain obscure, in relation to contributing chemicals. OBJECTIVES: To obtain more clarity about the chemicals that drive the deterioration of semen quality, we conducted a mixture risk assessment based on European exposures. METHODS: We included chemicals capable of affecting semen quality after prenatal exposures, among them androgen receptor antagonists, substances that disrupt prostaglandin signalling, suppress testosterone synthesis, inhibit steroidogenic enzymes or activate the aryl hydrocarbon receptor. We employed the Hazard Index approach (HI), based on risk quotients of exposures in Europe and reference doses for reductions in semen quality. By summing up the risk quotients of the 29 chemicals included in the assessment we examined fold-exceedances of "acceptable" mixture exposures relative to an index value of 1. For bisphenols A, F, S, phthalates DEHP, DnBP, BBzP, DiNP, n-butyl paraben and paracetamol we relied on biomonitoring studies in which these 9 chemicals were measured together in the same subjects. This allowed us to construct personalised Hazard Indices. RESULTS: Highly exposed subjects experienced combined exposures to the 9 chemicals that exceeded the index value of 1 by more than 100-fold; the median was a 17-fold exceedance. Accounting for median background exposures to the remaining 20 chemicals added a Hazard Index of 1.39. Bisphenol A made the largest contribution to the HI, followed by polychlorinated dioxins, bisphenols S and F and DEHP. Eliminating bisphenol A alone would still leave unacceptably high mixture risks. Paracetamol is also a driver of mixture risks among subjects using the drug. CONCLUSIONS: Tolerable exposures to substances associated with deteriorations of semen quality are exceeded by a large margin. Bisphenols, polychlorinated dioxins, phthalates and analgesics drive these risks. Dedicated efforts towards lowering exposures to these substances are necessary to mitigate risks.

Declining semen quality and polybrominated diphenyl ethers (PBDEs): Review of the literature to support the derivation of a reference dose for a mixture risk assessment.

To support a mixture risk assessment for chemicals that interfere with male reproductive health, we reviewed the literature to identify studies of polybrominated diphenyl ethers (PBDEs) and poor semen quality. Several epidemiological studies have shown associations of PBDE exposures with declining semen quality, non-descending testes and penile malformations. In rodent studies, poor semen quality, changes in testosterone levels and reproductive tissues have been observed. In vitro studies with reporter gene constructs show PBDE congeners as androgen receptor antagonists, and mixture studies in these systems have demonstrated that PBDE congeners act together with other androgen receptor antagonists. These observations led us to attempt the estimation of reference doses for specific PBDE congeners that can be used in a future mixture risk assessment for deteriorations of semen quality. While epidemiological studies provide support for such associations, they were uninformative for derivations of reference doses, due to the incompatibility of dose metrics used in exposure assessments. We therefore based our estimates on animal studies. Using a rigorous confidence rating approach, we found robust evidence that BDE-47 produced reductions in semen quality. We identified only one high confidence study of BDE-99 and accordingly evaluated the strength of evidence as moderate. One high confidence, and several medium confidence experimental studies observed declines in semen quality after BDE-209 exposure. Using established risk assessment procedures, we estimated that BDE-47 exposures below 0.15 µg/kg/d are unlikely to lead to reductions in semen quality. The corresponding exposures for BDE-99 and BDE-209 are 0.003 µg/kg/d and 1000 µg/kg/d. It is planned to use these estimates as reference doses in a mixture risk assessment of deteriorations in semen quality, involving multiple other chemicals also contributing to poor semen quality.

Bisphenol A and declining semen quality: A systematic review to support the derivation of a reference dose for mixture risk assessments.

To support a mixture risk assessment with a focus on male reproductive health, we conducted a systematic review of associations between bisphenol A (BPA) exposures and declines in semen quality, based on animal and epidemiological studies. Contrary to a widely held view that there is "conflicting" evidence of such associations, our review and confidence rating approach reveals that animal studies provide convincing evidence of declines of semen quality after gestational BPA exposures. Many of the reported negative findings can be attributed to deficiencies in study sensitivity, insufficient control of background contamination and probable confounding through hormonal interference due to the use of soy-containing diets. We did not evaluate animal studies of adult BPA exposures. Divergent findings in "medium to high" and "medium" confidence epidemiological studies can be explained in terms of differences in exposure conditions. We attempted the estimation of a BPA reference dose based on animal studies. Due to variations in the no-observed adverse effect levels (NOAELs) in high confidence studies, possible reference doses ranged from 0.0001 to 0.0099 µg/kg/d. In choosing 0.003 µg/kg/d we struck a balance between caution suggested by studies at the lower end of the doses and the weight of evidence from studies with higher NOAELs. This weighting was motivated by the intended use of the value in a mixture risk assessment which meant arriving at a reasonable estimate of BPA exposures likely without effects on semen quality. We realise that our approach does not conform with the standards necessary for deriving tolerable daily intakes (TDIs) for single chemical exposures, which is not our interest here. BPA exposures currently experienced by European populations and beyond are in excess of 0.003 µg/kg/d and even fall in the range where some epidemiological studies observed effects on semen quality as a result of BPA exposures in adulthood.

Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30-70 % for pre-adipocyte proliferation; this was 40-80 % for triglyceride accumulation induced by pyraclostrobin, 80-100 % for tributyltin, and 80-100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30-70 % active determinations for pyraclostrobin, 30-50 % for tributyltin, and 20-40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.

Ten years of research on synergisms and antagonisms in chemical mixtures: A systematic review and quantitative reappraisal of mixture studies.

BACKGROUND: Several reviews of synergisms and antagonisms in chemical mixtures have concluded that synergisms are relatively rare. However, these reviews focused on mixtures composed of specific groups of chemicals, such as pesticides or metals and on toxicity endpoints mostly relevant to ecotoxicology. Doubts remain whether these findings can be generalised. A systematic review not restricted to specific chemical mixtures and including mammalian and human toxicity endpoints is missing. OBJECTIVES: We conducted a systematic review and quantitative reappraisal of 10 years' of experimental mixture studies to investigate the frequency and reliability of evaluations of mixture effects as synergistic or antagonistic. Unlike previous reviews, we did not limit our efforts to certain groups of chemicals or specific toxicity outcomes and covered mixture studies relevant to ecotoxicology and human/mammalian toxicology published between 2007 and 2017. DATA SOURCES, ELIGIBILITY CRITERIA: We undertook searches for peer-reviewed articles in PubMed, Web of Science, Scopus, GreenFile, ScienceDirect and Toxline and included studies of controlled exposures of environmental chemical pollutants, defined as unintentional exposures leading to unintended effects. Studies with viruses, prions or therapeutic agents were excluded, as were records with missing details on chemicals' identities, toxicities, doses, or concentrations. STUDY APPRAISAL AND SYNTHESIS METHODS: To examine the internal validity of studies we developed a risk-of-bias tool tailored to mixture toxicology. For a subset of 388 entries that claimed synergisms or antagonisms, we conducted a quantitative reappraisal of authors' evaluations by deriving ratios of predicted and observed effective mixture doses (concentrations). RESULTS: Our searches produced an inventory of 1220 mixture experiments which we subjected to subgroup analyses. Approximately two thirds of studies did not incorporate more than 2 components. Most experiments relied on low-cost assays with readily quantifiable endpoints. Important toxicity outcomes of relevance for human risk assessment (e.g. carcinogenicity, genotoxicity, reproductive toxicity, immunotoxicity, neurotoxicity) were rarely addressed. The proportion of studies that declared additivity, synergism or antagonisms was approximately equal (one quarter each); the remaining quarter arrived at different evaluations. About half of the 1220 entries were rated as "definitely" or "probably" low risk of bias. Strikingly, relatively few claims of synergistic or antagonistic effects stood up to scrutiny in terms of deviations from expected additivity that exceed the boundaries of acceptable between-study variability. In most cases, the observed mixture doses were not more than two-fold higher or lower than the predicted additive doses. Twenty percent of the entries (N = 78) reported synergisms in excess of that degree of deviation. Our efforts of pinpointing specific factors that predispose to synergistic interactions confirmed previous concerns about the synergistic potential of combinations of triazine, azole and pyrethroid pesticides at environmentally relevant doses. New evidence of synergisms with endocrine disrupting chemicals and metal compounds such as chromium (VI) and nickel in combination with cadmium has emerged. CONCLUSIONS, LIMITATIONS AND IMPLICATIONS: These specific cases of synergisms apart, our results confirm the utility of default application of the dose (concentration) addition concept for predictive assessments of simultaneous exposures to multiple chemicals. However, this strategy must be complemented by an awareness of the synergistic potential of specific classes of chemicals. Our conclusions only apply to the chemical space captured in published mixture studies which is biased towards relatively well-researched chemicals. SYSTEMATIC REVIEW REGISTRATION NUMBER: The final protocol was published on the open-access repository Zenodo and attributed the following digital object identifier, doi: https://doi.org//10.5281/zenodo.1319759 (https://zenodo.org/record/1319759#.XXIzdy7dsqM).

Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders.

BACKGROUND: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed. OBJECTIVES: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. METHODS: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression. RESULTS: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (λ-cyhalothrin, pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions. DISCUSSION: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

The GOLIATH Project: Towards an Internationally Harmonised Approach for Testing Metabolism Disrupting Compounds.

The purpose of this project report is to introduce the European "GOLIATH" project, a new research project which addresses one of the most urgent regulatory needs in the testing of endocrine-disrupting chemicals (EDCs), namely the lack of methods for testing EDCs that disrupt metabolism and metabolic functions. These chemicals collectively referred to as "metabolism disrupting compounds" (MDCs) are natural and anthropogenic chemicals that can promote metabolic changes that can ultimately result in obesity, diabetes, and/or fatty liver in humans. This project report introduces the main approaches of the project and provides a focused review of the evidence of metabolic disruption for selected EDCs. GOLIATH will generate the world's first integrated approach to testing and assessment (IATA) specifically tailored to MDCs. GOLIATH will focus on the main cellular targets of metabolic disruption-hepatocytes, pancreatic endocrine cells, myocytes and adipocytes-and using an adverse outcome pathway (AOP) framework will provide key information on MDC-related mode of action by incorporating multi-omic analyses and translating results from in silico, in vitro, and in vivo models and assays to adverse metabolic health outcomes in humans at real-life exposures. Given the importance of international acceptance of the developed test methods for regulatory use, GOLIATH will link with ongoing initiatives of the Organisation for Economic Development (OECD) for test method (pre-)validation, IATA, and AOP development.

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer.

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple-negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three-dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6-depleted, triple-negative MDA-MB-231 cells rearranged the actin cytoskeleton and restored epidermal growth factor-mediated invasion. In patients with localized, lymph node-negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse-free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6-fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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.

Mind the gap: can we explain declining male reproductive health with known antiandrogens?

Several countries have experienced rises in cryptorchidisms, hypospadias and testicular germ cell cancer. The reasons for these trends are largely unknown, but Skakkebaek has proposed that these disorders form a testicular dysgenesis syndrome and can be traced to androgen insufficiency in foetal life. This suggests that antiandrogenic chemicals might contribute to risks, but few chemicals have been linked to these diseases in epidemiological studies. In animal studies with p,p'-dichlorodiphenyldichloroethylene, effects typical of disruptions of male sexual differentiation became apparent when the foetal levels of this androgen receptor (AR) antagonist approached values associated with responses in in vitro assays. This prompted us to analyse whether the 22 chemicals with AR antagonistic properties would produce mixture effects in an in vitro AR antagonism assay when combined at concentrations found in human serum. Other antiandrogenic modalities could not be considered. Two scenarios were investigated, one representative of average serum levels reported in European countries, the other in line with levels towards the high exposures. In both situations, the in vitro potency of the 22 selected AR antagonists was too low to produce combined AR antagonistic effects at the concentrations found in human serum, although the high exposure scenario came quite close to measurable effects. Nevertheless, our analysis exposes an explanation gap which can only be bridged by conjuring up as yet undiscovered high potency AR antagonists or, alternatively, high exposures to unknown agents of average potency.

Genotoxic mixtures and dissimilar action: concepts for prediction and assessment.

Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive.

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.

Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro.

Benzimidazoles act by disrupting microtubule polymerisation and are capable of inducing the formation of micronuclei. Considering the similarities in their mechanisms of action (inhibition of microtubule assembly by binding to the colchicine-binding site on tubulin monomers), combination effects according to the principles of concentration addition might occur. If so, it is to be expected that several benzimidazoles contribute to micronucleus formation even when each single one is present at or below threshold levels. This would have profound implications for risk assessment, but the idea has never been tested rigorously. To fill this gap, we analysed micronucleus frequencies for seven benzimidazoles, including the fungicide benomyl, its metabolite carbendazim, the anthelmintics albendazole, albendazole oxide, flubendazole, mebendazole and oxibendazole. Thiabendazole was also tested but was inactive. We used the cytochalasin-blocked micronucleus assay with CHO-K1 cells according to OECD guidelines, and employed an automated micronucleus scoring system based on image analysis to establish quantitative concentration-response relationships for the seven active benzimidazoles. Based on this information, we predicted additive combination effects for a mixture of the seven benzimidazoles by using the concepts of concentration addition and independent action. The observed effects of the mixture agreed very well with those predicted by concentration addition. Independent action underestimated the observed combined effects by a large margin. With a mixture that combined all benzimidazoles at their estimated threshold concentrations for micronucleus induction, micronucleus frequencies of ~15.5% were observed, correctly anticipated by concentration addition. On the basis of independent action, this mixture was expected to produce no effects. Our data provide convincing evidence that concentration addition is applicable to combinations of benzimidazoles that form micronuclei by disrupting microtubule polymerisation. They present a rationale for grouping these chemicals together for the purpose of cumulative risk assessment.

The suitability of concentration addition for predicting the effects of multi-component mixtures of up to 17 anti-androgens with varied structural features in an in vitro AR antagonist assay.

The risks associated with human exposures to chemicals capable of antagonising the effects of endogenous androgens have attracted considerable recent interest. Exposure is typically to large numbers of chemicals with androgen receptor (AR) antagonist activity, yet there is limited evidence of the combined effects of multi-component mixtures of these chemicals. A few in vitro studies with mixtures of up to six AR antagonists suggest that the concept of concentration addition (CA) provides good approximations of experimentally observed mixture effects, but studies with larger numbers of anti-androgens, and with more varied structural features, are missing. Here we show that the mixture effects of up to 17 AR antagonists, comprising compounds as diverse as UV-filter substances, parabens, perfluorinated compounds, bisphenol-A, benzo(α)pyrene, synthetic musks, antioxidants and polybrominated biphenyls, can be predicted well on the basis of the anti-androgenicity of the single components using the concept of CA. We tested these mixtures in an in vitro AR-dependent luciferase reporter gene assay, based on MDA-kb2 cells. The effects of further mixtures, composed of four and six anti-androgens, could be predicted accurately by CA. However, there was a shortfall from expected additivity with a ten-component mixture at two different mixture ratios, but attempts to attribute these deviations to differential expression of hormone-metabolising CYP isoforms did not produce conclusive results. CA provides good approximations of in vitro mixture effects of anti-androgens with varying structural features.

Joint effects of heterogeneous estrogenic chemicals in the E-screen--exploring the applicability of concentration addition.

In the last few years, significant advances have been made toward understanding the joint action of endocrine disrupting chemicals (EDCs). A number of studies have demonstrated that the combined effects of different types of EDCs (e.g., estrogenic, antiandrogenic, or thyroid-disrupting agents) can be predicted by the model of concentration addition (CA). However, there is still limited information on the effects of mixtures of large numbers of chemicals with varied structural features, which are more representative of realistic human exposure scenarios. The work presented here aims at filling this gap. Using a breast cancer cell proliferation assay (E-Screen), we assessed the joint effects of five mixtures, containing between 3 and 16 estrogenic agents, including compounds as diverse as steroidal hormones (endogenous and synthetic), pesticides, cosmetic additives, and phytoestrogens. CA was employed to predict mixture effects, which were then compared with experimental outcomes. The effects of two of the mixtures tested were additive, being accurately predicted by CA. However, for the three other mixtures, CA slightly overestimated the experimental observations. In view of these results, we hypothesized that the deviations were due to increased metabolism of steroidal estrogens in the mixture setting. We investigated this by testing the impact of two such mixtures on the activation and expression of steroidal estrogen metabolizing enzymes, such as cytochrome P450 (CYP) 1A1, CYP 1B1, and CYP 3A4. Activation of CYP 1B1 and, consequently, a reduction in the levels of steroidal estrogens in the mixture could contribute to the shortfall from the additivity prediction that we observed.

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.

Cell cycle-dependent 3D distribution of telomeres and telomere repeat-binding factor 2 (TRF2) in HaCaT and HaCaT-myc cells.

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.