Article

Something From “Nothing”-Eight Weak Estrogenic Chemicals Combined at Concentrations Below NOECs Produce Significant Mixture Effects

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Abstract

We tested whether multicomponent mixtures of xenoestrogens would produce significant effects when each component was combined at concentrations below its individual NOEC or EC01 level. The estrogenic effects of eight chemicals of environmental relevance, including hydroxylated PCBs, benzophenones, parabenes, bisphenol A, and genistein, were recorded using a recombinant yeast estrogen screen (YES). To ensure that no chemical contributed disproportionately to the overall combination effect, a mixture was prepared at a mixture ratio proportional to the potency of each individual component. The performance of four approaches for the calculation of additive combination effects (concentration addition, toxicity equivalency factors, effect summation, and independent action) was compared. Experimental testing of the predictions revealed that concentration addition and its application, the toxicity equivalency factor approach, were valid methods for the calculation of additive mixture effects. There was excellent agreement between prediction and observation. In contrast, independent action and effect summation led to clear underestimations of the experimentally observed responses. Crucially, there were substantial mixture effects even though each chemical was present at levels well below its NOEC and EC01. We conclude that estrogenic agents are able to act together to produce significant effects when combined at concentrations below their NOECs. Our results highlight the limitations of the traditional focus on the effects of single agents. Hazard assessments that ignore the possibility of joint action of estrogenic chemicals will almost certainly lead to significant underestimations of risk.

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... The disease burden and high costs (Tables 1 and 2) associated with individual EDCs represent only a narrow subset of the broader implications of EDCs for human health. Chemical mixtures, or compounds made up of two or more chemical components that are not necessarily chemically linked, can produce cumulative effects greater than those predicted by their individual constituent chemicals alone in both in vitro and in vivo models [94][95][96][97][98] . Combinations of chemicals at doses or concentrations that alone have low or no activity, for example, can produce additive or synergistic effects and/or can modulate the effects of background hormone activity 94,95 . ...
... Chemical mixtures, or compounds made up of two or more chemical components that are not necessarily chemically linked, can produce cumulative effects greater than those predicted by their individual constituent chemicals alone in both in vitro and in vivo models [94][95][96][97][98] . Combinations of chemicals at doses or concentrations that alone have low or no activity, for example, can produce additive or synergistic effects and/or can modulate the effects of background hormone activity 94,95 . Increasing research has also begun to evaluate the effects of chemical mixtures, and the adverse effects of more complex mixtures are being detected 99,100 , but >80% of mixture studies still currently focus on small, technically simple mixtures of two or three similar components 101 . ...
... If chemicals have been demonstrated to display additive and/or synergistic effects, this finding should trigger requirements for the consideration of co-exposure in the risk assessments for any chemical use. Finally, to adequately protect humans from harmful EDC exposure, health assessments should account for real-world mixture and cumulative exposures, particularly for exposure to chemicals acting through similar mechanisms of action 94,95,137 . ...
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Endocrine-disrupting chemicals (EDCs) are substances generated by human industrial activities that are detrimental to human health through their effects on the endocrine system. The global societal and economic burden posed by EDCs is substantial. Poorly defined or unenforced policies can increase human exposure to EDCs, thereby contributing to human disease, disability and economic damage. Researchers have shown that policies and interventions implemented at both individual and government levels have the potential to reduce exposure to EDCs. This Review describes a set of evidence-based policy actions to manage, minimize or even eliminate the widespread use of these chemicals and better protect human health and society. A number of specific challenges exist: defining, identifying and prioritizing EDCs; considering the non-linear or non-monotonic properties of EDCs; accounting for EDC exposure effects that are latent and do not appear until later in life; and updating testing paradigms to reflect 'real-world' mixtures of chemicals and cumulative exposure. A sound strategy also requires partnering with health-care providers to integrate strategies to prevent EDC exposure in clinical care. Critical next steps include addressing EDCs within global policy frameworks by integrating EDC exposure prevention into emerging climate policy.
... It is also important to note that OELs have limitations, including the fact that they may not be protective of workers during vulnerable periods such as the preconception or prenatal period. For example, there is some evidence to suggest that VOCs like benzene, ethylbenzene, and toluene are reproductive and developmental toxicants posing a potential risk to hairdressers during these sensitive periods [65] ; however, current OELs for these VOCs are based on acceptable risks derived from limited toxicological data rather than just developmental and reproductive toxicity [66,67] . Moreover, OELs may not protect workers with pre-existing chronic conditions such as asthma and do not account for the potential effects that may arise from chemical mixtures [66][67][68] . ...
... For example, there is some evidence to suggest that VOCs like benzene, ethylbenzene, and toluene are reproductive and developmental toxicants posing a potential risk to hairdressers during these sensitive periods [65] ; however, current OELs for these VOCs are based on acceptable risks derived from limited toxicological data rather than just developmental and reproductive toxicity [66,67] . Moreover, OELs may not protect workers with pre-existing chronic conditions such as asthma and do not account for the potential effects that may arise from chemical mixtures [66][67][68] . ...
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Hairdressers are exposed to volatile organic compounds (VOCs) that can pose health risks. Women of color (Black/Latina) represent nearly one-third of all U.S. hairdressers who may be disproportionally exposed to VOCs through occupational and personal use of hair products and treatments specifically formulated for this demographic. Still, data on workplace VOC exposures in this workforce remains sparse. We conducted area air monitoring of 14 VOCs in three salons serving Black women (“Black salons”), three Dominican salons predominantly serving Latino and Black women and 10 office spaces using active integrated sampling across 8-hour work shifts. Most VOCs measured were detected in hair salons (n = 13) and offices (n = 11). Salons had median VOC concentrations 2-175 times higher than offices. Among salons, 95th percentile VOC concentrations were up to 187 times higher in Black salons than in Dominican salons, suggesting that elevated exposures may occur partly from differences based on product use, services rendered, and salon characteristics (e.g., cleaning practices, ventilation). This is the first study to report indoor air concentrations of multiple individual targeted VOCs in U.S. hair salons serving women of color, highlighting the need for comprehensive exposure studies and assessment of potential health risks in this understudied and overexposed workforce.
... However, the fact that the toxicity of a single compound is known does not guarantee its behavior when combined with other substances. Compounds that share a common mechanism of action may be able to cooperate with each other and induce a measurable effect even if they are individually present below their No Observed Adverse Effect Level-NOAEL (Silva et al., 2002). These joint effects therefore complicate risk assessment, hence the need to extend current methods of toxicological assessment (Perkins et al., 2019). ...
... The experimental results were compared to the activity predicted by models derived from CA and IA concepts, to test whether the mixtures followed the additivity principle or showed signs of interaction. Mixtures have also been prepared containing compounds below their activity threshold to determine if the overall mixture activity was significant, illustrating the so-called "something out of nothing" paradigm (Silva et al., 2002). ...
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Introduction: Many natural or synthetic compounds used in foods, dietary supplements, and food contact materials (FCMs) are suspected endocrine disruptors (EDs). Currently, scientific evidence to predict the impacts on biological systems of ED mixtures is lacking. In this study, three classes of substances were considered: i) phytoestrogens, ii) plant protection products (PPP) and iii) substances related to FCMs. Fourteen compounds were selected based on their potential endocrine activity and their presence in food and FCMs. Methods: These compounds were evaluated using an in vitro gene expression assay, the ERα-CALUX, to characterize their responses on the estrogen receptor alpha. Cells were exposed to fixed ratio mixtures and non-equipotent mixtures of full and partial agonists. The concentration-response curves measured for the three classes of compounds were characterized by variable geometric parameters in terms of maximum response (efficacy), sensitivity (slope) and potency (median effective concentration EC50). To account for these variations, a generic response addition (GRA) model was derived from mass action kinetics. Results: Although GRA does not allow us to clearly separate the concentration addition (CA) and independent action (IA) models, it was possible to determine in a statistically robust way whether the combined action of the chemicals in the mixture acted by interaction (synergy and antagonism) or by additive behavior. This distinction is crucial for assessing the risks associated with exposure to xenoestrogens. A benchmark dose approach was used to compare the response of phytoestrogen blends in the presence and absence of the hormone estradiol (E2). At the same time, 12 mixtures of 2–5 constituents including phytoestrogens, phthalates and PPPs in proportions close to those found in food products were tested. In 95% of cases, the response pattern observed showed a joint and independent effect of the chemicals on ER. Discussion: Overall, these results validate a risk assessment approach based on an additive effects model modulated by intrinsic toxicity factors. Here, the CA and IA approaches cannot be distinguished solely based on the shape of the concentration response curves. However, the optimized GRA model is more robust than CA when the efficacy, potency, and sensitivity of individual chemical agonists show large variations.
... Organisms in these ecosystems are often exposed to a complex mixture of chemical pollutants. This exposure can sometimes lead to harmful effects, even when the concentrations of individual pollutants are below their No Observable Effect Concentration (NOEC) (Brian et al., 2007;Kortenkamp, 2008;Silva et al., 2002). This phenomenon, known as cocktail effects, combined effects, mixture toxicity, or joint toxicity, underscores the complexity of chemical interactions. ...
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This chapter explores the multifaceted threats posed by global warming to agriculture, the foundation of human civilization. It argues that climate change is not a singular threat, but rather a complex web of challenges, including rising temperatures, erratic precipitation patterns, water scarcity, increased pests and diseases, and declining crop nutrition. The chapter highlights how these threats disproportionately impact developing countries. However, the chapter also offers a ray of hope. It discusses various adaptation strategies that can be employed to build resilience in agricultural systems, such as breeding heat-tolerant crops, utilizing smarter water management practices, embracing precision agriculture techniques, adopting Integrated Pest Management (IPM), and integrating trees into agricultural landscapes through agroforestry practices. The chapter concludes by emphasizing the need for a collective effort to address this challenge. It calls for robust government policies, international cooperation, and investment in research and development to ensure a sustainable and food-secure future for all.
... Although there are policies regarding safe levels of exposure of EDCs, these regard single chemicals and not exposure to mixtures of EDCs, which correspond to real-life exposures where humans always are exposed to a huge number of compounds [2]. While it is important to study single chemical exposures as previous studies have done [12,13], mixture approaches should be studied in more detail, as there might be a joint action between chemicals where the adversity is not present in single compound analysis but can be detected in mixture analyses; i.e., the something from nothing phenomenon [19,20]. ...
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Background Endocrine disrupting chemicals (EDCs) can cross the placenta and thereby expose the fetus, which may lead to developmental consequences. It is still unclear which chemicals are of concern regarding neurodevelopment and specifically behaviour, when being exposed to a mixture. Objective The objective is to determine associations between prenatal exposure to EDCs and behavioural difficulties. Furthermore, we investigated sex-specific associations and determined chemicals of concern in significant regressions. Methods Associations between prenatal exposure to EDCs (both as single compounds and their mixtures) and behavioural outcomes using the Strengths and Difficulties Questionnaire (SDQ) were estimated in 607 mother-child pairs in the Swedish Environmental Longitudinal, Mother and Child, Asthma and Allergy (SELMA) study. Levels for chemical compounds were measured in either urine or serum (median of 10 weeks of gestation). Associations were estimated for the total SDQ score (quasipoisson regression) and a 90th percentile cut-off (logistic regression). Exposure for EDC mixtures (phenols, phthalates, PFAS and persistent chlorinated) was studied using weighted quantile sum (WQS) regression with deciles and with and without repeated holdout validation techniques. The models were adjusted for selected covariates. Results The odds for behavioural difficulties increased in girls with higher chemical exposures (OR 1.77, 95% CI 1.67, 1.87) using the full sample and borderline for the validation set (OR 1.31, 95% CI 0.93, 1.85) with 94/100 positive betas in the 100 repeated holdout validations. Chemicals of concern for girls are mostly short-lived chemicals and more specifically plasticizers. No pattern of significant associations was detected for boys. Significance There is an indication of increased behavioural difficulties for girls in the SELMA population with higher exposure to mixtures of EDCs. Using the repeated holdout validation techniques, the inference is more stable, reproducible and generalisable. Prenatal exposure to mixtures of environmental chemicals should be considered when assessing the safety of chemicals. Impact Growing evidence points towards a “mixture effect” where different environmental chemicals might act jointly where individual compounds may be below a level of concern, but the combination may have an effect on human health. We are constantly exposed to a complicated mixture pattern that is individual for every person as this mixture depends on personal choices of lifestyle, diet and housing to name a few. Our study suggests that prenatal exposure to EDCs might adversely affect the behaviour of children and especially girls. Hence, risk assessment needs to improve and sex-specific mechanisms should be included in assessments.
... Zn plays a pivotal role in more than 300 biological processes [38], and an imbalance in Zn homeostasis is associated with neurological and psychiatric illnesses such as Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder and depression [39]. Studies have shown that Zn deficiency impairs spontaneous behavior and exacerbates dopaminergic neuronal death [40], whereas Zn supplementation improves spatial memory and attenuates neuronal apoptosis [41]. In our study, Zn supplementation improved learning impairments in nematodes and attenuated the apoptosis of HT22 cells. ...
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Environmental metal mixtures can cause combined neurotoxicity, but the underlying mechanism remains unclear. Mitochondria are crucial for energy metabolism in the nervous system, and their dysfunction leads to neurodegeneration. Zinc (Zn) is a coenzyme of many mitochondrial enzymes that controls mitochondrial function. This study investigated the role of Zn in the neurotoxicity induced by Mn + Pb and Pb + As mixtures. Zn supplementation improved the survival rate and learning ability of Caenorhabditis elegans following their exposure to mixtures of Mn + Pb and Pb + As by enhancing their mitochondrial morphology, membrane potential, and respiratory chain. Similarly, in HT22 cells, Zn mitigated the decrease in cellular activity and increase in apoptosis induced by the Mn + Pb and Pb + As mixtures by improving mitochondrial morphology and function. Mechanistically, Zn activated the PINK1 and MFN-2/OPA-1 pathways, promoting mitophagy and mitochondrial fusion. However, inhibition of mitophagy reversed the protective effect of Zn, indicating its reliance on mitophagy for neuroprotection. Our study demonstrated that Zn alleviates the combined neurotoxicity of Mn + Pb and Pb + As mixtures by enhancing mitophagy and mitochondrial fusion, suggesting that Zn supplementation is a potential treatment for metal-induced neurotoxicity. Graphical Abstract
... Most epidemiological and experimental studies have focused on correlating single exposures to EDCs with a specific outcome. However, a principle of "something from nothing" has been described, by which EDC mixtures can provoke biologically relevant alterations at levels that single chemicals do not induce measurable effects [14]. Thus, investigating the combined effect of EDC mixtures on health and reproduction is physiologically relevant and could provide a more accurate estimation of EDC exposure risk. ...
Article
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Endocrine disruptor chemicals (EDCs) are natural and synthetic compounds found ubiquitously in the environment that interfere with the hormonal-immune axis, potentially impacting human health and reproduction. Exposure to EDCs has been associated with numerous health risks, such as neurodevelopmental disorders, metabolic syndrome, thyroid dysfunction, infertility, and cancers. Nevertheless, the current approach to establishing causality between EDCs and disease outcomes has limitations. Epidemiological and experimental research on EDCs faces challenges in accurately assessing chemical exposure and interpreting non-monotonic dose response curves. In addition, most studies have focused on single chemicals or simple mixtures, overlooking complex real-life exposures and EDC mechanistic insights, in particular regarding their impact on the immune system. The ENDOMIX project, funded by the EU’s Horizon Health Program, addresses these challenges by integrating epidemiological, risk assessment, and immunotoxicology methodologies. This systemic approach comprises the triangulation of human cohort, in vitro, and in vivo data to determine the combined effects of EDC mixtures. The present review presents and discusses current literature regarding human reproduction in the context of immunotolerance and EDC mode of action. It further underscores the ENDOMIX perspective to elucidate the impact of EDCs on immune-reproductive health.
... Two distinct patterns emerged indicating mixture effects with interaction between components. In the first pattern, which was revealed with moderate effect size for the variables synchrony of germination (Z; G. mollugo, S. latifolia) and germination percentage (GP; G. mollugo), the mixture showed a significant effect compared to the control, while the individual components did not, termed as a "something from "nothing"" response (Silva et al., 2002). However, in only one case (Z, S. latifolia) was there a significant difference between the MIX treatment and one of the single treatments. ...
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Seed germination and early growth of grassland species might be influenced by veterinary antibiotics that are extensively released into agricultural habitats. Therefore, we tested impacts of the commonly used antibiotics tetracycline and sulfamethazine, single and in mixture, on seed germination and seedling root growth of six typical species of temperate European grasslands (Carum carvi, Centaurea jacea, Galium mollugo, Plantago lanceolata, Silene latifolia, Dactylis glomerata). In standardised germination experiments, we assessed three germination variables (germination percentage, mean germination time, synchrony of germination) and one post-germination variable (seedling root length) under different environmentally realistic antibiotic concentrations (0.1, 1, 10 mg l-1 and a water control). While the germination variables were only irregularly and weakly affected by both antibiotics, seedling root length was strongly reduced by tetracycline, but not by sulfamethazine. Among the test species, D. glomerata was most sensitive to tetracycline with the average root length reduced up to 81 % in the 10 mg l-1 treatment. Its germination behaviour, however, was almost insensitive to the two antibiotics. Mixture effects were only shown in relation to the germination of single species, where the binary mixture produced effects but not the two single antibiotics or, conversely, effects of single antibiotics were lost in the mixture. These findings highlight the potential threat of plant regeneration from seed by veterinary antibiotics, particularly affecting early root growth and potentially influencing plant population growth in natural habitats.
... However, in nature, aquatic organisms are affected by complex mixtures of contaminants and continuously higher temperatures. This is relevant since the effects of mixtures of estrogenic compounds [35,36] and even of compounds with different mechanisms of action [37] can be more detrimental than those of the same compounds in single exposures. In line with this, additive (estrogenic) effects of EE2 and LNG on the CYP19A1B gene expression in the brain were reported in zebrafish (Danio rerio), in vivo and in vitro [38]. ...
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Fish are exposed to increased water temperatures and aquatic pollutants, including endocrine-disrupting compounds (EDCs). Although each stressor can disturb fish liver metabolism independently, combined effects may exist. To unveil the molecular mechanisms behind the effects of EDCs and temperature, fish liver cell lines are potential models needing better characterisation. Accordingly, we exposed the rainbow trout RTL-W1 cells (72 h), at 18 °C and 21 °C, to ethynylestradiol (EE2), levonorgestrel (LNG), and a mixture of both hormones (MIX) at 10 µM. The gene expression of a selection of targets related to detoxification (CYP1A, CYP3A27, GST, UGT, CAT, and MRP2), estrogen exposure (ERα, VtgA), lipid metabolism (FAS, FABP1, FATP1), and temperature stress (HSP70b) was analysed by RT-qPCR. GST expression was higher after LNG exposure at 21 °C than at 18 °C. LNG further enhanced the expression of CAT, while both LNG and MIX increased the expressions of CYP3A27 and MRP2. In contrast, FAS expression only increased in MIX, compared to the control. ERα, VtgA, UGT, CYP1A, HSP70b, FABP1, and FATP1 expressions were not influenced by the temperature or the tested EDCs. The RTL-W1 model was unresponsive to EE2 alone, sensitive to LNG (in detoxification pathway genes), and mainly insensitive to the temperature range but had the potential to unveil specific interactions.
... Even chemicals with low intrinsic hazard may be transformed by environmental conditions (such as sunlight 42 or pyrolysis 43 ) into more toxic products 36 . Moreover, even when individual chemicals are present below levels of concern, their complex interactions within chemical mixtures can induce toxicity [44][45][46][47][48] , as shown for some endocrine disruptors 47,49 . ...
... Nevertheless, existing researches have concentrated on the effects of a speci c class of chemicals on MetS [19,21], failing to clarify the association of mixed exposure to multiple classes of chemicals with MetS. In addition, simultaneous exposure to various chemicals may result in signi cant combined effects owing to the synergy or addiction effects of the chemical components, although an individual chemical exposure may not result in observable consequences [22]. Therefore, multiple combined exposure models are imperative to accurately calculate the integrated impacts of chemical combinations. ...
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Background Pesticides, polycyclic aromatic hydrocarbons (PAHs), and phthalates are recognized as potential contributors to metabolic disorders. Nevertheless, the combined effect of simultaneous exposure to these chemicals on the MetS remains elusive. Objectives To explore the impacts of simultaneous exposure to three classes of chemicals and identify critical chemicals. Methods Based on the NHANES database from 2007–2012, our study included 4,030 non-pregnant individuals aged 20 years or older. We used the weighted linear regression model, variable selection models (including LASSO regression and BMA models), as well as the mixture exposure model (WQS model) to investigate the correlation between chemicals and MetS. Additionally, stratified analyses were performed based on gender and age. Results The weighted generalized linear regression model revealed a positive correlation of 2-PHEN with MetS (OR: 1.37, 95% CI: 1.19–1.59, P < 0.001). Both the LASSO regression and BMA models identified 2-PHEN as a significant chemical positively associated with MetS. Additionally, the WQS model showed a positive association between overall exposure to the three chemical categories and MetS, with the highest weighted chemicals being 2-PHEN, MEOHP 2-NAP, and 2,5-DCP. Stratified analyses demonstrated a significant correlation between 2-PHEN and MetS between different subgroups in the weighted generalized linear regression, LASSO regression, and BMA models. Notably, the WQS regression model revealed a significant association in the subgroup of female (OR = 1.40, 95% CI: 1.08–1.83, P < 0.05), with 2-PHEN, 2,5-DCP, 2-NAP, and MEP identified as the primary contributions to Mets. Conclusion Combined exposure to the three chemical groups was positively related to a higher risk of MetS, with the PAH group exhibiting the most pronounced effect and 2-PHEN emerging as a key chemical with consistent and robust findings, promoting public health concerns regarding the potential health hazards of EDCs exposure to metabolic diseases.
... Furthermore, the mixed toxic effects of exposure to multiple BPs may differ significantly from those of individual BPs. For example, BPA exposure along with seven other estrogenic chemicals exhibited significant estrogenic activity while each chemical was at a concentration below its effect threshold [36]. Therefore, future research should be more focused on improving the accuracy of risk evaluations and the continued regulation of occupational exposure to BPs. ...
Article
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Electronic waste (e-waste) dismantling facilities are well-known bisphenol chemical (BP) sources. In this study, non-targeted screening combined with targeted analysis of BPs in surface soil from e-waste dismantling facilities and their surroundings revealed their presence, distribution, and exposure risk. A total of 14 BPs were identified including bisphenol A (BPA) and its novel structural analogs and halogenated BPs. The total concentrations of BPs ranged from 963 to 47,160 ng/g (median: 6970 ng/g) in e-waste soil, higher than those measured in surface soil from surrounding areas, i.e., 10–7750 ng/g (median 197 ng/g). BPA, tetrabromobisphenol A (TBBPA), and bisphenol F (BPF) were the dominant ones from the two areas. Concentrations of TBBPA and its debromination product from the surrounding area significantly decreased with increasing distances from the e-waste dismantling facilities. Estimation of daily intake via oral ingestion of soil suggests that current contamination scenarios are unlikely to pose health risks for e-waste dismantling workers and adults and toddlers living in the surrounding areas, with their intakes generally well below the tolerable daily intakes proposed for several BPs. However, the BPA intakes of workers exceeded the more strict tolerable daily intake for BPA established recently, which merits continuous environmental surveillance.
... There is a wealth of monitoring studies that analysed surface waters, wastewater treatment plant effluents, marine and freshwater or biota with targeted chemical screening approaches revealing the co-occurrence of up to 2000 chemicals as mixtures in the environment (e.g., [26,27]). At the same time, there is clear evidence from laboratory studies that even if all chemicals in a mixture are present at concentrations that do not cause adverse effects, i.e., that do not exceed their individual No Observed Effect Concentrations (NOECs), the mixture might still cause significant toxicity (e.g., [14,[28][29][30][31]. Additionally, several studies describe mixture risks for the environment from real exposure situations through a combination of measured or modelled concentrations of chemicals with the respective toxicity data (e.g., [7,9,[32][33][34][35][36]). ...
Article
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There is indisputable evidence that the environment, humans and wildlife are continuously exposed not to single but to multiple chemicals from different sources. Exposure to these mixtures can lead to combined risks not yet sufficiently addressed in any of the European chemical legislations. Under the REACH regulation for industrial chemicals, specific environmental mixture assessments are challenged by a lack of data on toxicity, use and exposures and the communication of data along the supply chain. Within the Chemicals Strategy for Sustainability the European Commission proposed to introduce (a) mixture allocation factor(s) (MAF) as regulatory management tool to reduce exposures, effects and potential risks of unintentional mixtures. The MAF is proposed to be applied as default value within the chemical safety assessments undertaken by companies under REACH. Here, we critically review the relevant literature discussing the conceptual background of the MAF and approaches to derive its magnitude. The analysis focuses on the environment and key issues for an implementation in regulatory practise together with remaining uncertainties and needs for possible ways forward. At this stage introducing a MAF in REACH Annex I appears the most pragmatic and immediately implementable measure to address risks from unintentional mixtures in the environment. A so-called MAFceiling appears as the preferred option of policy makers, since it would only affect relevant substances close to their respective risk threshold. While the magnitude of a MAF will be decided politically, the choice of methods and assumptions to derive its size should be clear and transparent, build on the available scientific evidence and take account for uncertainties. A MAF will be most effective reducing environmental releases and exposure levels if risk mitigation measures are implemented in practise. Its socioeconomic impacts and costs need to be assessed in a balanced way together with the benefits for the environment, society, and for companies—also in comparison to the efforts needed for specific mixture risk assessments. In the future and with the experiences gathered in practise, a discussion is needed on how to assess and regulate unintentional mixtures across different pieces of chemicals legislation to consider the true exposure situation and ensure harmonisation.
... As a traditional water quality testing method, targeted chemical analysis is unlikely to capture all the chemicals, especially at low concentrations, and may ignore the possible combinatorial effects of complex mixtures of pollutants. For example, while some individual pollutants are weakly toxic at environmental levels and their health risks are negligible, the presence of their mixtures can have adverse effects on ecosystems and humans and their health risks can no longer be ignored [9,10]. A battery of in vitro bioassays have emerged as a sensitive and reliable method to determine the toxic or endocrine-disrupting effects of individual chemicals or mixtures of all active chemicals in environmental samples [11,12]. ...
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The safety of drinking water is a significant environmental issue of great concern for human health since numerous contaminants are often detected in drinking water and its sources. Boiling is a common household method used to produce relatively high-quality drinking water in some countries and regions. In this study, with the aid of an integrated approach of in vitro bioassays and non-target analysis based on high-resolution mass spectrometry coupled with liquid chromatography, alterations in endocrine-disrupting activities in tap water samples without and with boiling were revealed, as well as the potential endocrine-disrupting chemicals (EDCs) contributing to these alterations were identified. The organic extracts of tap water had no significant (ant)agonistic activities against an estrogen receptor (ER), progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) at enrichment concentrations of ≤10 times, posing no immediate or acute health risk to humans. However, the presence of agonistic activities against PR and MR and antagonistic activities against ER, PR, GR, and MR in OEs of tap water at relatively higher enrichment concentrations still raise potential health concerns. Boiling effectively reduced antagonistic activities against these steroid hormone receptors (SHRs) but increased estrogenic and glucocorticoid activities in drinking water. Four novel potential EDCs, including one UV filter (phenylbenzimidazole sulfonic acid, PBSA) and three natural metabolites of organisms (beta-hydroxymyristic acid, 12-hydroxyoctadecanoic acid, and isorosmanol) were identified in drinking water samples, each of which showed (ant)agonistic activities against different SHRs. Given the widespread use of UV filters in sunscreens to prevent skin cancer, the health risks posed by PBSA as an identified novel EDC are of concern. Although boiling has been thought to reduce the health risk of drinking water contamination, our findings suggest that boiling may have a more complex effect on the endocrine-disrupting activities of drinking water and, therefore, a more comprehensive assessment is needed.
... For many years, the field of toxicology focused on understanding how individual chemicals affect biological systems. However, it has become clear in recent years that individual chemicals can have a cumulative effect: exposure to safe levels of multiple chemicals can be toxic in aggregate [1,2]. Even worse, durable "forever" chemicals accumulate in the environment and contribute to the growing list of chemicals people are exposed to every day [3]. ...
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Environmental toxicants overwhelmingly occur together as mixtures. The variety of possible chemical interactions makes it difficult to predict the danger of the mixture. In this work, we propose the novel Reflected Generalized Concentration Addition (RGCA), a piece-wise, geometric technique for sigmoidal dose-responsed inverse functions that extends the use of generalized concentration addition (GCA) for 3+ parameter models. Since experimental tests of all relevant mixtures is costly and intractable, we rely only on the individual chemical dose responses. Additionally, RGCA enhances the classical two-step model for the cumulative effects of mixtures, which assumes a combination of GCA and independent action (IA). We explore how various clustering methods can dramatically improve predictions. We compare our technique to the IA, CA, and GCA models and show in a simulation study that the two-step approach performs well under a variety of true models. We then apply our method to a challenging data set of individual chemical and mixture responses where the target is an androgen receptor (Tox21 AR-luc). Our results show significantly improved predictions for larger mixtures. Our work complements ongoing efforts to predict environmental exposure to various chemicals and offers a starting point for combining different exposure predictions to quantify a total risk to health.
... Although this toxicity equivalent conversion based on the concentration addition model 82 has been widely used in the study of various pollutants [84][85][86] and makes it possible to assess the combined toxicity of multiple herbicides in natural seawater, it is also necessary to admit that it may introduce some bias due to the varying susceptibility of different species to herbicides. To minimize this effect, we selected the most representative species possible for the singlesubstance concentration-response experiments, as mentioned above. ...
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Predicting the magnitude of herbicide impacts on marine primary productivity remains challenging because the extent of worldwide herbicide pollution in coastal waters and the concentration-response relationships of phytoplankton communities to multiple herbicides are unclear. By analyzing the spatiotemporal distribution of herbicides at 661 bay and gulf stations worldwide from 1990 to 2022, we determined median, third quartile and maximum concentrations of 12 triazine herbicides of 0.18 nmol L−1, 1.27 nmol L−1 and 29.50 nmol L−1 (95%Confidence Interval: CI 1.06, 1.47), respectively. Under current herbicide stress, phytoplankton primary productivity was inhibited by more than 5% at 25% of the sites and by more than 10% at 10% of the sites (95%CI 3.67, 4.34), due to the inhibition of highly abundant sensitive species, community structure/particle size succession (from Bacillariophyta to Dinophyceae and from nano-phytoplankton to micro-phytoplankton), and resulting growth rate reduction. Concurrently, due to food chain cascade effects, the dominant micro-zooplankton population shifted from larger copepod larvae to smaller unicellular ciliates, which might prolong the transmission process in marine food chain and reduce the primary productivity transmission efficiency. As herbicide application rates on farmlands worldwide are correlated with residues in their adjacent seas, a continued future increase in herbicide input may seriously affect the stability of coastal waters.
... Studien zur Quantifizierung der hormonellen Wirksamkeit verschiedener Derivate berichten außerdem von relativ hohen Effektkonzentrationen, die in dieser Messkampagne in keiner der Proben nachgewiesen werden konnten. Dennoch muss davon ausgegangen werden, dass vor allem hydroxylierte benzophenonbasierte UV-Filter auch bei vergleichsweise geringen Konzentrationen zu einer Gesamtöstrogenität natürlicher Gewässer beitragen können Kunz and Fent, 2006;Silva et al., 2002). Hierzu zählt auch das Transformationsprodukt von BP, 4-OH-BP, dessen PNEC-Wert mit 80 ng/L in einem sehr niedrigen Bereich liegt (Guo et al., 2020). ...
... The term micropollutants refers to a variety of chemical compounds such as pesticides, pharmaceuticals, personal care products and industrial chemicals, which are widely distributed as a result of diffuse pollution or insufficient wastewater treatment and can have direct toxic effects on various aquatic organisms (Malaj et al., 2014;Posthuma et al., 2020;Bradley et al., 2021;Halbach et al., 2021). In the environment, micropollutants are present in complex mixtures, i.e., spatially and temporally variable combinations of multiple compounds that may contribute to a joint mixture toxicity even if each individual compound occurs at non-toxic concentration (Silva et al., 2002;Escher et al., 2020). Micropollutants and their mixtures negatively affect aquatic ecosystems at different biological endpoints, e.g., survival and reproduction of sensitive species, taxonomic composition and biodiversity (Malaj et al., 2014;Mor et al., 2019;Posthuma et al., 2020;Liess et al., 2021). ...
... Concentrations of the tested MP were below the LOEC determined for sea urchin (LOEC MPP635-XF = 100 mg/L, LOEC Aquatex-325 = 100 mg/L) and mussel (LOEC MPP635-XF >100 mg/L) (Beiras et al., 2018). However, it is possible that the leachates have additive effects to the dissolved pollutant, which would help to explain the increase in toxicity observed for MP + Pol treatments (Bellas, 2008;Silva et al., 2002). In fact, although the products used here are considered 'virgin' MPs, the occurrence of low concentrations of different compounds such as plastic additives and PAHs (e.g. ...
Article
The objective of this study was to determine whether and to what extent microplastics (MPs) enhance the toxicity of pollutants as well as whether pollutant-loaded MPs act as relevant vectors of chemical pollutants. With this aim, the toxicity for mussel and sea urchin embryos of: 1) three dissolved pollutants (Pol): chlorpyrifos (CPF), fluoranthene (FLT) and mercury (Hg); 2) their mixture with Microplastics (MP + Pol); and 3) pollutant-loaded MPs (MPPol), was assessed. Analyses of CPF, FLT and Hg were also performed to evaluate the transfer among dissolved and particulate phases. In general, the 'MP + Pol' treatments were more toxic as 48-h EC50 (μg/L) than the 'Pol' treatments for sea urchin or mussel. The 48-h and 120-h EC50s (μg/L) for sea urchin showed little variation for CPF and MP + CPF, and no clear pattern was found for FLT and MP + FLT. The performed chemical analysis in the MPPol tests indicated that desorption was the main route to explain the observed toxicity of Hg and a relevant route for CPF and FLT. This study contributes to improve the knowledge about the interactions between MPs and chemical pollutants, which is fundamental for a more realistic ecological risk assessment in aquatic ecosystems.
... However, the methods are challenged by missing information on human exposure, toxicity, and possible unknown mixture components, such as emerging chemicals [5,6]. Furthermore, synergistic or antagonistic interactions among the chemicals may be overlooked [1,7]. ...
Article
Humans are simultaneously exposed to complex chemical mixtures, and its combined effect can affect human health. As part of the HBM4EU project, the actual mixture of perfluoroalkylated substances (PFAS) in 25 human placenta samples was extracted by chromatographic methods and assessed for xeno-estrogenic activity using two in-vitro bioassays: the estrogen receptor transactivity and the E-Screen. Most of the PFAS extracts displayed xeno-estrogenic activity, in one or both assays. The xeno-estrogenic activities in the two bioassays were not correlated, but both assays showed an overall negative correlation with placenta concentrations of single PFAS. Xeno-estrogenic activities were significantly related to maternal characteristics; being higher in young, smokers and primiparous women, but not with fetal growth (birth weight, birth length, head circumference, gestational age, placenta weight). The presented extraction method can be used to study the combined effect of real-life mixtures of PFAS in relation to health outcomes in large-scale human biomonitoring studies.
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Background Freshwater fish are important food sources that also pose risks to human and wildlife health because of the bioaccumulation of environmental chemicals in their tissues. Although most studies, fish consumption advisories, and regulations focus on individual contaminants, fish consumers are exposed to mixtures of chemicals, including legacy contaminants and contaminants of emerging concern, that can have combined effects. Chemicals of emerging concern represent one source of hazard, but legacy contaminants can still pose threats to fish consumers due to their persistence in the environment. Objectives We investigate the following questions: 1) Do different chemicals correlate with one another in fish tissue, and if so, how? 2) How do levels of different chemicals in fish tissue vary by time and location? and 3) How do observed chemical levels compare with risk-based screening levels? Methods Using several national data sources established and maintained by the US Environmental Protection Agency (NRSA, NCCA-GL, GLENDA, and NLFTS), this study examines the co-occurrence of chemicals in freshwater fish in lakes, ponds, streams, and rivers in the US. Results We determine that organic contaminants correlate with one another, but generally not with mercury; organic chemicals have declined over time, but mercury has not; and fish concentrations of legacy contaminants—even those banned for decades—continue to exceed risk-based screening levels. Discussion Despite some successes in curtailing release of pollutants, some contaminants in fish tissue have not declined and legacy and emerging pollutants continue to pose risks to fish consumers in the US. Correlations between chemicals in fish tissue suggest that exposures to mixtures is prevalent in the US but that organic contaminants do not generally correlate with mercury—noteworthy particularly since fish consumption advisories in the US are frequently driven by the level of mercury, and do not account for exposure to multiple contaminants. While programs such as the National Aquatic Resource Surveys (NARS) Program seek to systematically monitor contaminants in fish tissue and other environmental indicators, continuous support from the US federal government is required to sustain this monitoring. Moreover, greater legislative and regulatory efforts are required at both the state and federal levels to reduce continuing sources and ongoing contamination.
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Organic and inorganic chemicals co-occur in household dust, and these chemicals have been determined to have endocrine and metabolic disrupting effects. While there is increasing study of chemical mixtures, the effects of complex mixtures mimicking household dust and other environmental matrices have not been well studied and their potential metabolism disrupting effects are thus poorly understood. Previous research has demonstrated high potency adipogenic effects of residential household dust extracts using in vitro adipogenesis assays. More recent research simplified this to a mixture relevant to household dust and comprised of common co-occurring organic and inorganic contaminants, finding that these complex combinations often exhibited additive or even synergistic effects in cell models. This study aimed to translate our previous in vitro observation to an in vivo model, the developing zebrafish, to evaluate the metabolic effects of early exposure to organic and inorganic chemicals, individually and in mixtures. Zebrafish embryos were exposed from 1 day post fertilization (dpf) to 6 dpf, then metabolic energy expenditure, swimming behavior and gene expression were measured. Globally, we observed that most mixtures did not reflect the effects of individual chemicals; the BFR mixture produced a less potent effect when compared to the individual chemicals, while the PFAS and the inorganic mixtures seemed to have a more potent effect than the individual chemicals. Finally, the environmental mixture, mimicking household dust proportions, was less potent than the inorganic chemical mix alone. Additional work is necessary to better understand the mixture effect of inorganic and organic chemicals combined.
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One of the most significant challenges in human health risk assessment is to evaluate hazards from exposure to environmental chemical mixtures. Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous contaminants typically found as mixtures in gaseous and particulate phases in ambient air pollution associated with petrochemicals from Superfund sites and the burning of fossil fuels. However, little is understood about how PAHs in mixtures contribute to toxicity in lung cells. To investigate mixture interactions and component additivity from environmentally relevant PAHs, two synthetic mixtures were created from PAHs identified in passive air samplers at a legacy creosote site impacted by wildfires. The primary human bronchial epithelial cells differentiated at the air–liquid interface were treated with PAH mixtures at environmentally relevant proportions and evaluated for the differential expression of transcriptional biomarkers related to xenobiotic metabolism, oxidative stress response, barrier integrity, and DNA damage response. Component additivity was evaluated across all endpoints using two independent action (IA) models with and without the scaling of components by toxic equivalence factors. Both IA models exhibited trends that were unlike the observed mixture response and generally underestimated the toxicity across dose suggesting the potential for non-additive interactions of components. Overall, this study provides an example of the usefulness of mixture toxicity assessment with the currently available methods while demonstrating the need for more complex yet interpretable mixture response evaluation methods for environmental samples.
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Organophosphate esters (OPEs) are used primarily as flame retardants and plasticizers. Previously, we reported that adrenal cells are important targets of individual OPEs. However, real life exposures are to complex mixtures of these chemicals. To address this, we exposed H295R human adrenal cells to varying dilutions (1/1000 K to 1/3 K) of a Canadian household dust based OPE mixture for 48 h and evaluated effects on phenotypic, lipidomic, and functional parameters. Using a high-content screening approach, we assessed phenotypic markers at mixture concentrations at which there was greater than 70% cell survival; the most striking effect of the OPE mixture was a 2.5-fold increase in the total area of lipid droplets. We then determined the response of specific lipid species to OPE exposures with novel, non-targeted lipidomic analysis of isolated lipid droplets. These data revealed that house dust OPEs induced concentration-dependent alterations in the composition of lipid droplets, particularly affecting the triglyceride, diglyceride, phosphatidylcholine, and cholesterol ester subclasses. The steroid producing function of adrenal cells in the presence or absence of a steroidogenic stimulus, forskolin, was determined. While the production of 17β-estradiol remained unaffected, a slight decrease in testosterone production was observed after stimulation. Conversely, a 2-fold increase in both basal and stimulated cortisol and aldosterone production was observed. Thus, exposure to a house dust based mixture of OPEs exerts endocrine-disrupting effects on adrenal cells, highlighting the importance of assessing the effects of environmentally relevant mixtures.
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The ecological hazard and risk assessment of individual chemicals in Japan have been far behind the other developed countries such as the US and European countries and have long been limited to fish acute toxicity assessment for pesticides. After the recommendation by Organization for Economic Cooperation and Development (OECD) in 2002, ecological hazard and risk assessment for industrial chemicals was implemented in Chemical Substances Control Law (CSCL) and that for pesticides was expanded to crustaceans and algae in 2003 by Ministry of the Environment. Environmental standard for water quality to protect aquatic organisms was also first implemented in 2003 for zinc while the guidance for the ecological risk assessment for human pharmaceuticals was implemented at last in 2016 by Ministry of Health, Labor, and Welfare. Due to the increasing number of chemicals manufactured, used, and wasted, the ecological hazard and risk assessment for complex mixtures have become concern and investigated but the component-based approach in the ecological risk assessment has not yet well established and is far behind that in human health risk assessment. Alternatively, whole mixture approach such as the direct bioassay of effluent and ambient water has been implemented in some developed countries but the attempt to implement the Japanese version of Whole Effluent Toxicity system was ended up with the voluntary-based measure. Therefore, component-based approach for the grouping of chemicals based on chemical structure, use and mode of action should be started by the development of guidance documents possibly used in CSCL and other regulations with utilizing new approach methods (NAMs) in addition to conventional ecological testing combined with proper whole mixture approach for monitoring effluent and ambient water to cover such growing numbers of diverse small production volume chemicals.
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Non-dioxin-like polychlorinated biphenyls (NDL PCBs) alter the activity of the ryanodine receptor (RyR), and this activity is linked to developmental neurotoxicity. Most work to date has focused on the activity of single congeners rather than relevant mixtures. The current study assessed the RyR activity of single congeners or binary, tertiary, and complex PCB mixtures. Observed mixture activity was then compared to the expected activity calculated using the concentration addition (CA) model or a RyR-specific neurotoxic equivalency scheme (rNEQ). The predictions of the CA model were consistent with the observed activity of binary mixtures at the lower portion of the concentration-response curve, supporting the additivity of RyR1 active PCBs. Findings also show that minimally active congeners can compete for the RyR1 binding site, and congeners that do not activate the RyR1 do not interfere with the activity of a full agonist. Complex PCB mixtures that mimic PCB profiles detected in indoor air, fish tissue, and the serum of mothers and children activated the RyR1 and displayed similar efficacy and potency regardless of varying congener profiles. Neither the CA model nor the rNEQ perfectly predicted the observed activity of complex mixtures, but predictions were often within one magnitude of change from the observed response. Importantly, PCB mixtures approximating profiles found in environmental samples or human serum displayed RyR1 activity at concentrations reported in published research. The work presented will aid in the development of risk assessment platforms for NDL PCBs, and similar compounds, towards RyR1 activation and related neurotoxicity.
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Bisphenol analogues (BPs) are ubiquitous in the environment and have gained significant attention regarding their associated health risks. However, there is a lack of comprehensive biomonitoring data on BPs and their metabolites in human urine. To address this, we conducted a study evaluate the exposure to BPs in the general population of Guangzhou, China. A total of 1440 urine samples were collected from volunteers and analyzed for the presence of BPs and their metabolites after being pooled into 36 groups based on age and gender. The findings revealed the common detection of ten free-form BPs, as well as the urinary metabolites of BPA and BPS, in the pooled urine samples. BPA was the predominant free-form compound, constituting 50% of the total BPs. The primary urinary metabolites of BPA and BPS are BPA-G and BPS-G, respectively, indicating glucuronidation as their primary metabolic pathway. The composition of urinary metabolites of BPA and BPS varied by age and sex, while the concentration of total BPs in urine was not significantly associated with age and sex. Enzymatic hydrolysis yielded a mean amplification of individual BPs concentrations in urine samples ranging from 1.8 times (BPA) to 4.6 times (BPS). Based on the outcomes, it was estimated that conjugated forms accounted for 96.9%, 96.2%, 94.7%, 94.1%, 92.6%, 89.1%, 87.3%, 87.2%, 87.1% and 85.8% of BPP, BPAF, BPZ, BPE, BPAP, BPF, BPA, BPC, BPS and BPF, respectively, in the pooled urine samples. Preliminary risk assessments indicated that the estimated daily intake of BPA was much higher than the latest proposed tolerable daily intake. Due to the unavailability of health-based guideline values for alternative BPs, some of them exhibit daily intakes comparable to BPA, implying that greater attention should be paid to health risks associated with exposure to BPs.
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We tested whether bisphenol A (BPA) or o,p'-DDT, when combined with 17beta-estradiol (E2), would contribute to the overall mixture effect using a yeast reporter gene assay, the yeast estrogen screen. Following comprehensive concentration-response analyses of the single agents, the pharmacologically well-founded models of concentration addition and independent action were used to predict entire concentration-response relationships for mixtures of the agents with a variety of fixed mixture ratios, assuming additivity. For molar mixture ratios proportional to the levels normally found in human tissues (i.e., below 1:5000, E2:BPA or o,p'-DDT), these predictions suggest that the effects of individual xenoestrogens are too weak to create an impact on the actions of steroidal hormones. However, at mixture ratios more in favor of the xenoestrogens, a significant contribution to the overall mixture effect was predicted. The predictions were tested experimentally. The observed combined effects of mixtures of E2 with either BPA or o,p'-DDT did not deviate from the additivity expectation. On combining E2 with either BPA or o,p'-DDT at approximately equieffective concentrations corresponding to molar mixture ratios between 1:20,000 and 1:100,000 (E2:BPA or o,p'-DDT), substantial modulations of the effects of E2 became discernible. The assumption that weak xenoestrogens are generally unable to create an impact upon the already strong effects of endogenous steroidal estrogens is not supported by our observations. Our studies indicate that the potential health implication of additive combination effects between xenoestrogens and steroidal estrogens deserve serious consideration.
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An estrogen-inducible screen was developed in yeast (Saccharomyces cerevisiae) in order to assess whether surfactants and their major degradation products are estrogenic. The DNA sequence of the human estrogen receptor (hER) was integrated into the yeast genome, which also contained expression plasmids carrying estrogen-responsive sequences (ERE) controlling the expression of the reporter gene lac-Z (encoding the enzyme β-galactosidase). Thus, in the presence of estrogens, β-galactosidase is synthesized and secreted into the medium, where it causes a color change from yellow to red. This recombinant strain was used to determine whether representatives of major surfactant classes and some of their principal degradation products possess estrogenic activity. The results were compared to the effects of the main natural estrogen 17β-estradiol. None of the parent surfactants tested possessed estrogenic activity. However, one class of surfactants, the alkylphenol polyethoxylates, degrade to persistent metabolites that were weakly estrogenic. Another group of degradation products, the sulfophenyl carboxylates, which are derived from the biodegradation of linear alkylbenzene sulfonates, do not appear to possess estrogenic activity.
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We have utilized a validated (standardized) estrogen receptor (ER) competitive-binding assay to determine the ER affinity for a large, structurally diverse group of chemicals. Uteri from ovariectomized Sprague-Dawley rats were the ER source for the competitive-binding assay. Initially, test chemicals were screened at high concentrations to determine whether a chemical competed with [3H]-estradiol for the ER. Test chemicals that exhibited affinity for the ER in the first tier were subsequently assayed using a wide range of concentrations to characterize the binding curve and to determine each chemical's IC 50 and relative binding affinity (RBA) values. Overall, we assayed 188 chemicals, covering a 1 × 106-fold range of RBAs from several different chemical or use categories, including steroidal estrogens, synthetic estrogens, antiestrogens, other miscellaneous steroids, alkylphenols, diphenyl derivatives, organochlorines, pesticides, alkylhydroxybenzoate preservatives (parabens), phthalates, benzophenone compounds, and a number of other miscellaneous chemicals. Of the 188 chemicals tested, 100 bound to the ER while 88 were non-binders. Included in the 100 chemicals that bound to the ER were 4-benzyloxyphenol, 2,4-dihydroxybenzophenone, and 2,2′-methylenebis(4-chlorophenol), compounds that have not been shown previously to bind the ER. It was also evident that certain structural features, such as an overall ring structure, were important for ER binding. The current study provides the most structurally diverse ER RBA data set with the widest range of RBA values published to date.
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The carcinogenic potentials of 40 National Toxicology Program chemicals previously predicted by Computer Optimised Molecular Parametric Analysis for Chemical Toxicity (COMPACT), based on the identification of potential substrates of cytochromes P4501A and 2E (CYP1A and CYP2E), have been compared with new rodent carcinogenicity results. The COMPACT predictions have also been compared with published Ames mutagenicity data and with our own Hazardexpert predictions for carcinogenicity. Concordance evaluations between rodent carcinogenicity (1/4 segments positive) and predictions by COMPACT or Hazardexpert were 64% for COMPACT (CYP1A only), 72% for COMPACT (CYP1A plus CYP2E), 70% for Hazardexpert alone, and 86% for COMPACT (CYP1A plus CYP2E) plus Hazardexpert. Sensitivities of the predictions were for COMPACT, 75%; Hazardexpert, 60%; and Ames, 54%. Positive predictivities were for COMPACT, 75%; Hazardexpert, 78%; and Ames 81%. Negative predictivites were for COMPACT, 62%; Hazardexpert, 52%; and Ames, 42%.
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The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.
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There is considerable public, regulatory, and scientific concern regarding human exposure to endocrine-disrupting chemicals, which include compounds that directly modulate steroid hormone receptor pathways (estrogens, antiestrogens, androgens, antiandrogens) and aryl hydrocarbon receptor (AhR) agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Based on quantitative structure-activity relationships for both AhR and estrogen receptor (ER) agonists, the relative potency (RP) of individual compounds relative to a standard (e.g. TCDD and 17-beta-estradiol) have been determined for several receptor-mediated responses. Therefore, the TCDD or estrogenic equivalent (TEQ or EQ, respectively) of a mixture is defined as TEQ = sigma[T(i)]xRP(i)or EQ=sigma[E(i)]xRP(i), where T(i) and E(i) are concentrations of individual AhR or ER agonists in any mixture. This approach for risk assessment of endocrine-disrupting mixtures assumes that for each endocrine response pathway, the effects of individual compounds are essentially additive. This paper will critically examine the utility of the TEQ/EQ approach for risk assessment, the validity of the assumptions used for this approach, and the problems associated with comparing low dose exposures to xeno and natural (dietary) endocrine disruptors.
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The assessment of mixture effects of estrogenic agents is regarded as an issue of high priority by many governmental agencies and expert decision-making bodies all over the world. However, the few mixture studies published so far have suffered from conceptual and experimental problems and are considered to be inconclusive. Here, we report the results of assessments of two-, three- and four-component mixtures of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol, all compounds with well-documented estrogenic activity. Extensive concentration-response analyses with the single agents were carried out using a recombinant yeast screen (yeast estrogen screen, YES). Based on the activity of the single agents in the YES assay we calculated predictions of entire concentration-response curves for mixtures of our chosen test agents assuming additive combination effects. For this purpose we employed the models of concentration addition and independent action, both well-established models for the calculation of mixture effects. Experimental concentration-response analyses revealed good agreement between predicted and observed mixture effects in all cases. Our results show that the combined effect of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol in the YES assay does not deviate from expected additivity. We consider both reference models as useful tools for the assessment of combination effects of multiple mixtures of xenoestrogens.
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We used a recombinant yeast estrogen assay to assess the activity of 73 phenolic additives that are used as sunscreens, preservatives, disinfectants, antioxidants, flavorings, or for perfumery. Thirty-two of these compounds displayed activity: 22 with potencies relative to 17beta-estradiol, ranging from 1/3,000 to < 1/3,000,000, and 10 compounds with an impaired response that could not be directly compared with 17beta-estradiol. Forty-one compounds were inactive. The major criteria for activity appear to be the presence of an unhindered phenolic OH group in a para position and a molecular weight of 140-250 Da.
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In view of the large differences between the concentrations of estrogenic chemicals needed to elicit effects in in vitro assays and their levels in human tissues, it is hard to explain possible health risks in terms of exposure to individual compounds. Human populations, however, are exposed to mixtures of estrogenic and estrogen-like agents and it is necessary to consider the impact of combined effects. We assessed the combined effects of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p'-DDT), 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), beta-hexachlorocyclohexane (beta-HCH), and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (p,p'-DDT) on the induction of cell proliferation in MCF-7 cells. All four compounds are persistent organochlorines that can be found in human tissues. We performed extensive concentration-response analyses with the single agents to predict the effects of two mixtures of all four compounds with different mixture ratios. We calculated the predictions by using the pharmacologically well-founded models of concentration addition and independent action and then tested them experimentally. o,p'-DDT, p,p'-DDE, beta-HCH, and p,p'-DDT acted together to produce proliferative effects in MCF-7 cells. The combined effect of the four agents could be predicted on the basis of data about single agent concentration-response relationships. Regression analysis demonstrated that there were combination effects even when each mixture component was present at levels at or below its individual no-observed-effect-concentration. We assessed combination effects in two ways: First, evaluations in relation to the proliferative responses induced by single mixture components revealed that the combination effects were stronger than the effects of the most potent constituent. Thus, according to this method of evaluation, the combined effects may be termed synergistic. Second, comparisons with the expected effects, as predicted by concentration addition and independent action, showed excellent agreement between prediction and observation. With this approach, the combined effect of all four compounds can be termed additive.
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This paper questions the usefulness of the no-observed-effect level (NOEL) as a summary statistic for ecotoxicological experiments. Quantification of the NOEL depends critically on size and variability of an experiment: smaller and less precise experiments lead to higher values for the NOEL, which gives the wrong signal to suppliers of a NOEL. The NOEL will generally lie in a dose range where possible effects cannot be excluded, even within the context of the experiment. Hence it does not form a suitable starting point for extrapolation to field situations. Alternative methods of finding a dose with a negligible effect are discussed. A two-step procedure is proposed that involves finding the dose whose effect is at most 25%, followed by linear extrapolation to a dose whose effect is acceptably small. The procedure leads to higher values if efforts are made to increase the power of the experiment.
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Two morphological types of tubers of yellow nutsedge (Cyperus esculentus L.) were collected over a 2-year period and were sprouted in the laboratory. Tuber dormancy occurred during late summer and early fall. Sprouting was highest during the winter and spring. Mechanical disturbance of the nutsedge stand increased tuber sprouting. Available carbohydrates followed a pattern similar to sprouting; minimum levels were found during late summer. The two types of tubers appeared to be similar in respect to the characteristics studied.
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The responses of herbicides applied singly are used in calculating the "expected" response when they are combined. The expected response for a combination is obtained by taking the product of the percent-of-control values for herbicides applied alone and dividing by (100)ⁿ⁻¹ where n is the number of herbicides in the combination.
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Some time ago, a multiple comparison procedure for comparing several treatments simultaneously with a control or standard treatment was introduced by the present author (Dunnett [1955]). The procedure was designed to be used either to test the significance of the differences between each of the treatments and the control with a stated value 1 - P for the joint significance level, or to set confidence limits on the true values of the treatment differences from the control with a stated value P for the joint confidence coefficient. Thus the procedure has the property of controlling the experimentwise, rather than the per-comparison, error rate associated with the comparisons, in common with the multiple comparison procedures of Tukey [unpublished] and Scheffe [1953]. In the earlier paper, tables were provided enabling up to nine treatments to be compared with a control with joint confidence coefficient either .95 or .99. Tables for both one-sided and two-sided comparisons were given but, as explained in the paper, the two-sided values were inexact for the case of more than two comparisons as a result of an approximation which had to be made in the computations. The main purpose of the present paper is to give the exact tables for making two-sided comparisons. The necessary computations were done on a General Precision LGP-30 electronic computer, by a method described in section 3 below. The tables are given here as Tables II and III; these replace Tables 2a and 2b, respectively, of the previous paper. In addition to providing the exact values, a method is given for adjusting the tabulated values to cover the situation where the variance of the control mean is smaller than the variance of the treatment means, as occurs for example when a greater number of observations is allocated to the control than to any of the test treatments. Furthermore, the number of treatments which may be simultaneously compared with a control has been extended to twenty. 482
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This paper questions the usefulness of the no-observed-effect level (NOEL) as a summary statistic for ecotoxicological experiments. Quantification of the NOEL depends critically on size and variability of an experiment: smaller and less precise experiments lead to higher values for the NOEL, which gives the wrong signal to suppliers of a NOEL. The NOEL will generally lie in a dose range where possible effects cannot be excluded, even within the context of the experiment. Hence it does not form a suitable starting point for extrapolation to field situations. Alternative methods of finding a dose with a negligible effect are discussed. A two-step procedure is proposed that involves finding the dose whose effect is at most 25%, followed by linear extrapolation to a dose whose effect is acceptably small. The procedure leads to higher values if efforts are made to increase the power of the experiment.
Article
The prediction of combined effects based on the effects of the individual components of mixtures by using the pharmacological concepts of concentration addition and independent action might be a promising tool for the risk assessment of pollutant mixtures. To analyze and compare the predictive capabilities of the reference concepts for similarly acting chemicals, the overall toxicity of a multiple mixture was determined in a bioluminescence inhibition assay with Vibrio fischeri. The mixture was composed of 16 similarly and specifically acting chemicals, anticipated to have a common mode of action via weak acid respiratory uncoupling of oxidative phosphorylation. Results show that the observed mixture toxicity is rather well predicted by both concepts. Concentration addition shows an excellent predictive power; the median effective concentration (EC50) of the mixture is predicted with an error of about 10%. Independent action, in contrast, underestimates the EC50 of the mixture by a factor of a little more than three. With respect to risk assessment procedures, it may be concluded that concentration addition gives a valid estimation of the overall toxicity for multiple mixtures with similar and specific mechanisms of action of the mixture components in this type of biotest.
Article
Chronic aquatic toxicity test results are commonly analyzed with statistical hypothesis tests to generate summary statistics known as the no-observed-effect concentration (NOEC) and first-observed-effect concentration (FOEC). These procedures address statistical differences among treatments but suffer several critical limitations. Use of concentration-response statistics to estimate minimal effect concentrations (i.e., EC values) from quantal and continuous data has advantages over a hypothesis-testing approach for generating a biologically relevant end point and an estimate of variability from toxicity tests. Estimation of the concentration-response statistic (EC, effective concentration) for continuous data is not straightforward but is possible with a variety of approaches. A statistical method for estimating EC values described here is based on a nonlinear regression estimation procedure. The usefulness of this method is demonstrated with continuous data from chronic toxicity tests with algae, fish, and invertebrate populations.
Article
Bliss's (1939) statistical treatment of the hypothesis of the independent joint action of poisons is extended and revised. The possibility of negative correlation in resistance is considered, and the bivariate normal correlation coefficient is used to measure correlation in resistance. There are interesting characteristics of the dosage-mortality curve for a mixture of poisons the resistances to which are log-normally distributed: these are described. The question of whether a mixture of poisons is more toxic if they act similarly or independently is discussed.
Article
It is now possible to predict toxicity of mixtures of two or more pollutants on the basis of chemical measurements. Toxicities of individual components are added up as fractions of the incipientlc50 (lethal threshold concentration). Total toxicity of the mixture is expressed as a single number. A standard terminology is given for the effects of two or more toxicants acting simultaneously.Modifying conditions (temperature, water hardness, etc.) greatly affect toxicity. Much fragmentary information should be brought together and examined for underlying patterns. Concise summaries for estimating modifying effects have been published for rainbow trout. Computerized multivariable analysis is a promising tool, especially for studies of sublethal toxicity. Effects of fluctuating concentrations of toxicants may be evaluated by four approximate techniques; the theoretical basis for understanding effects still seems unproven. Acclimation to toxicants should be studied in detail for representative pollutants.There is a great deal of information on relative resistance of different species of fish but it needs summarizing. A trend towards use of ‘standard’ fish species in research is good. With invertebrates and algae, as much as with fish, tests of sublethal effects such as growth and development are often more meaningful than tests of acute toxicity.Chemical autopsy methods seem promising, more so than histopathological approaches, which suffer from practical difficulties in field situations.RésuméMaintenant il est possible de prédire la toxicité d'un mélange de deux ou plusieurs polluants sur la base de mesurages chimiques. Les toxicités des composants individuels sont additionnées comme fractions du naissantlc50 (seuil de concentration léthale). La toxicité totale du mélange est exprimée comme un seul nombre. Une terminologie standard est donnée pour les effets de deux ou plusieurs toxiques agissant simultanément.Des conditions modifiantes (température, dureté de l'eau, etc.) affèctent grandement la toxicité. Plus d'informations fragmentaires devraient être assemblées et examinées pour des critères fondamentaux. Des résumés concis pour l'évaluation des effets modifiants ont été publiés pour la truite... (arc-en-ciel). L'analyse multivariable par ordinateur est un outil prometteur, surtout pourl'étude de la toxicité sous-léthale. Les effects des concentrations fluctuantes des toxiques peuvent être évalués par quatre techniques approximatives; la base théorique pour la compréhension des effets reste toujours non-éprouvée. L'acclimatation aux toxiques devrait être étudiée en détail pour les polluants représentatifs.Il y a beaucoup d'information sur la résistance relative de différentes espèces de poissons, mais il est nécessaire de la résumer. Une tendance vers l'utilisation des espèces de poissons “standard” est bonne. Avec les invertébrés et les algues autant qu'avec les poissons, les tests des effets sous-léthals, comme la croissance et le développement, sont souvent plus significatifs que les tests de toxicité aigue.Les méthodes d'autopsie chimique paraissent prometteuses, même plus que les abords histopathologiques, qui souffrent à cause de difficultés pratiques dans les situations sur le terrain.ZusammenfassungMan ist heutezutage imstande, Toxizität in Gemischen bestehend aus zwei oder mehreren Verunreinigern auf Grundlage von chemischen Messungen vorauszusagen. Die Toxizitäten von einzelnen Bestandteilen werden als Fraktionen des Anfangsstadiums “lc50” (Todesschwellenkonzentration) zusammengezählt. Vollständige Toxizität einer Mischung wird als eine einzige Zahl ausgedrückt. Es wird eine Standardterminologie für die Wirkungen von zwei oder mehreren Giftstoffen, die gleichzeitig in Aktion treten, aufgestellt.Modifizierende Umstände (wie Temperatur, Die Härte des Wassers, u.s.w.) haben grossen Einfluss auf die Toxizität. Es sollte viel fragmentarische Information zusammengetragen und auf zugrundeliegende Beispielsformen untersucht werden. Prägnante Zusammenfassungen über die Schätzung der modifizierenden Effekte wurden über die Regenbogenforelle veröffentlicht. Durch Komputer erzielte, sehr abwandlungsfähige Analysen sind vielversprechend, besonders für Studien über sublethale Toxizität. Die Wirkung von fluktuierenden Giftstoffkonzentrationen kann mittels vier approximativen Methoden geschätzt werden; die theore tische Grundlage zum Verständnis der Wirkung scheint noch immer unbewiesen. Akklimatisierung an Giftstoffe sollte genauest an repräsentativen Verunreinigern studiert werden.Es liegt viel Information über den relativen Widerstand von verschiedenen Fischarten vor; diese muss jedoch zusammengefasst werden. Der Trend, für Forschung “Standard”-Fischarten zu verwenden, ist gut. Bei wirbellosen Tieren und Algen sowie auch bei Fischen sind Tests über die sublethale Wirkung, wie Wuchs und Entwicklung, oft vielbedeutender als Tests über akute Toxizität.Chemische Autopsiemethoden scheinen vielversprechend, besser als histopathologische Angriffsmethoden, die bei Aussenstudien praktischen Schwierigkeiten unterliegen.
Article
There are concerns about possible combination effects of environmental chemicals with oestrogenic activity and their implications for human health. Such chemicals are present in complex mixtures in our environment. A number of studies searching for possible synergistic interactions between xenoestrogens have appeared in the literature. However, in these studies no account was taken of established concepts and methods for analysing combination effects. In the present review, we highlight conceptual issues which may be useful for a sound analysis of the effects of mixtures of xenoestrogens. We find that much published work suffers from an undue focus on measuring effects of mixtures at only one dose level. Assessments of combination effects are frequently complicated by a lack of information on dose–response relationships. Some studies which purportedly show absence of synergy have in fact overlooked synergisms.
Article
A promising tool for the risk assessment of chemical mixtures is the prediction of their toxicities from the effects of the individual components. For that purpose, concentration addition is uniformly regarded as valid for mixtures of similarly acting chemicals. Whether this concept or the competing notion of independent action is more appropriate for mixtures of dissimilarly acting chemicals is still in dispute. Therefore, the presented study analyzed and compared the predictive capabilities of both concepts for a multiple mixture designed of strictly dissimilarly acting compounds. Experimental investigations were conducted using a long-term bioluminescence inhibition assay with Vibrio fischeri. Results show an excellent predictive power of independent action, while concentration addition overestimates the mixture toxicity. Thus, the precise prediction of mixture toxicities depends on a valid assessment of the similarity/dissimilarity of the mixture components. However, concentration addition underestimates the EC50 of the mixture only by a factor of less than three. As the similarity of components is often unknown for mixtures found in the environment, it is concluded that concentration addition may give a realistic worst case estimation of mixture toxicities for risk assessment procedures.
Article
The assessment of the combined effects of substances is usually based on one of two different concepts: concentration addition or independent action. Both concepts are founded on different pharmacological assumptions about sites and modes of actions of substances, but in toxicology and ecotoxicology such knowledge is rare for most chemicals. In order to validate experimental results and to allow for precautions assessments, the quantitative relationships between concentration addition and independent action are therefore of interest. In this paper, we derive for the Weibull, the logistic, and the normal distribution functions the concentrations where the response probability due to concentration addition exceeds that due to independent action and vice versa. This is done (a) by analytically comparing both models for low and high mixture concentrations and (b) by numerically calculating the response probabilities when concentration addition and independent action agree. It is shown that the relationships between the models for joint action depend on the distribution functions, the corresponding slope parameters, and on the mixture concentrations administered.
Article
Halogenated aromatic compounds, typified by the polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and diphenylethers (PCDEs), are industrial compounds or byproducts which have been widely identified in the environment and in chemical-waste dumpsites. Halogenated aromatics are invariably present in diverse analytes as highly complex mixtures of isomers and congeners and this complicates the hazard and risk assessment of these compounds. Several studies have confirmed the common receptor-mediated mechanism of action of toxic halogenated aromatics and this has resulted in the development of structure-activity relationships for this class of chemicals. The most toxic halogenated aromatic is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and based on in vivo and in vitro studies the relative toxicities of individual halogenated aromatics have been determined relative to TCDD (i.e., toxic equivalents). The derived toxic equivalents can be used for hazard and risk assessment of halogenated aromatic mixtures; moreover, for more complex mixtures containing congeners for which no standards are available (e.g., bromo/chloro mixtures), several in vitro or in vivo assays can be utilized for hazard or risk assessment.
Article
Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.
Article
I write to formally withdraw the report “Synergistic activation of estrogen receptor with combinations of environmental chemicals” ([7 June 1996, p. 1489][1]) ([1][2]), for which I was corresponding author. We have conducted experiments duplicating the conditions of our earlier work, but have
Article
Chemicals that act as androgen receptor (AR) agonists and antagonists or inhibit fetal steroidogenesis can induce reproductive malformations in humans and laboratory animals. Several environmental chemicals disrupt development in rats and/or rabbits at fetal concentrations at, or near, exposure levels seen in some segments of the human population. In rats, fetal tissues concentrations of 10-20 p.p.m. of the DDT metabolite, p,p'-DDE, are correlated with reproductive abnormalities in male offspring. These concentrations are similar to those measured in first-trimester human fetal tissues in the late 1960s. The pesticides vinclozolin, procymidone, linuron and DDT are AR antagonists. They reduce male rat anogenital distance, and induce areolas at relatively low dosages. Hypospadias, agenesis of the sex accessory tissues and retained nipples are seen in the middle dosages, while undescended testes and epididymal agenesis are seen in the highest doses. Phthalate esters (PE) inhibit testosterone synthesis during fetal life, but do not appear to be AR antagonists. Prenatal administration of a single low dose of dioxin (50-1,000 ng TCDD/kg) alters the differentiation of androgen-dependent tissues at p.p.t. concentrations, but the mechanism of action likely involves interaction with a hormone-like nuclear transcription factor, the hormone-like receptor AhR, rather than AR. p,p'-DDT and p,p'-DDE, vinclozolin and di-n-butyl phthalate affect reproductive function in rabbits when administered during prenatal and/or neonatal life. Cryptorchidism and carcinoma in situ-like (CIS) testicular lesions were seen in male rabbits treated during development with p,p'-DDT or p,p'-DDE. Extrapolation of effects from rodents to humans would be enhanced if future studies incorporate determination of tissue concentrations of the active metabolites. Knowledge of the tissue concentrations of the active toxicants also would provide an important link to in-vitro studies, which provide more useful mechanistic information when they are executed at relevant concentrations.
Article
Experiments were conducted to assess the in vivo potency of binary mixtures of estrogenic chemicals using plasma vitellogenin (VTG) concentrations in juvenile rainbow trout (Oncorhynchus mykiss) as the endpoint. The estrogenic potencies of estradiol-17beta (E2), 4-tertnonylphenol (NP), and methoxychlor (MXC) were determined following 14 day exposures to the individual chemicals and binary mixtures of these chemicals. E2, NP, and MXC all induced concentration dependent increases in plasma VTG, with lowest observed effect concentrations of 4.7 and 7.9 ng L(-1) for E2, 6.1 and 6.4 microg L(-1) for NP, and 4.4 and 6.5 microg L(-1) for MXC. Concentration-response curves for fixed ratio binary mixtures of E2 and NP (1:1000), E2 and MXC (1:1000), and NP and MXC (1:1) were compared to those obtained for the individual chemicals, using the model of concentration addition. Mixtures of E2 and NP were additive at the concentrations tested, but mixtures of E2 and MXC were less than additive. This suggests that while NP probably acts via the same mechanism as E2 in inducing VTG synthesis, MXC may be acting via a different mechanism(s), possibly as a result of its conversion to HPTE which is an estrogen receptor alpha agonist and an estrogen receptor beta antagonist. It was not possible to determine whether mixtures of MXC and NP were additive using VTG induction, because the toxicity of MXC restricted the effect range forwhich the expected response curve forthe binary mixture could be calculated. The data presented illustrate that the model of concentration addition can accurately predict effects on VTG induction, where we know that both chemicals act via the same mechanism in mediating a vitellogenic response.
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