Mixtures of Estrogenic Chemicals Enhance Vitellogenic Response in Sea Bass

BACKGROUND: The potential impact of natural and synthetic estrogens on aquatic ecosystems has attracted considerable attention because it is currently accepted that their joint effects are more severe when they are present in mixtures. Although it is well-known that they occur as mixtures in the marine environment, there is little information about the combined effects of estrogenic chemicals on marine biota. OBJECTIVE: In 14-day tests with juvenile sea bass, we analyzed singly and in combination the estrogenic activity of estradiol (E2), ethynylestradiol (EE2), and bisphenol A (BPA) using vitellogenin induction as an end point. METHODS: Fish were exposed to each compound, and on the basis of these concentration-response data, we predicted mixture effects by applying the model of concentration addition. The mixtures were tested using a fixed-ratio design, and the resulting mixture effects were compared to the predictions. RESULTS: EE2 was the most potent steroid, with an EC50 (median effective concentration) of 0.029 μg/L, 3.6 times more potent than E2 (EC50 = 0.104 μg/L); BPA was the least potent chemical, with an EC50 of 77.94 μg/L. The comparative assessment yielded a good agreement between observed and predicted mixture effects. CONCLUSIONS: This study demonstrates the potential hazard of these compounds to seawater life by their ability to act together in an additive manner. It provides evidence that concentration addition can be used as a predictive tool for assessing the combined effects of estrogenic chemicals in marine ecosystems

Analysis of Lipid Metabolism, Immune Function, and Neurobehavior in Adult C57BL/6JxFVB Mice After Developmental Exposure to di (2-ethylhexyl) Phthalate

Background: Developmental exposure to di (2-ethylhexyl) phthalate (DEHP) has been implicated in the onset of metabolic syndrome later in life. Alterations in neurobehavior and immune functions are also affected by phthalate exposure and may be linked to the metabolic changes caused by developmental exposure to DEHP.Objectives: Our goal was to study the effects of developmental exposure to DEHP in the context of metabolic syndrome by integrating different parameters to assess metabolic, neurobehavioral, and immune functions in one model.Methods: Female C57BL/6J mice were exposed to DEHP through the diet during gestation and lactation at doses ranging from 3.3 to 100,000 μg/kg body weight/day (μkd). During a 1-year follow-up period, a wide set of metabolic parameters was assessed in the F1 offspring, including weekly body weight measurements, food consumption, physical activity, glucose homeostasis, serum lipids, and endocrine profile. In addition, neurobehavioral and immune functions were assessed by sweet preference test, object recognition test, acute phase protein, and cytokines production. Animals were challenged with a high fat diet (HFD) in the last 9 weeks of the study.Results: Increased free fatty acids (FFA) and, high density lipoprotein (HDL-C) were observed in serum, together with a decrease in glycated hemoglobin levels in blood of 1-year old male DEHP-exposed offspring after HFD challenge. For the most sensitive endpoint measured (FFA), a lower bound of the 90%-confidence interval for benchmark dose (BMD) at a critical effect size of 5% (BMDL) of 2,160 μkd was calculated. No persistent changes in body weight or fat mass were observed. At 33,000 μkd altered performance was found in the object recognition test in males and changes in interferon (IFN)γ production were observed in females.Conclusions: Developmental exposure to DEHP combined with HFD in adulthood led to changes in lipid metabolism and neurobehavior in male offspring and cytokine production in female offspring. Our findings contribute to the evidence that DEHP is a developmental dyslipidemic chemical, however, more research is needed to further characterize adverse health outcomes and the mechanisms of action associated with the observed sex-specific effects

The role of analytical chemistry in exposure science: Focus on the aquatic environment

Exposure science, in its broadest sense, studies the interactions between stressors (chemical, biological, and physical agents) and receptors (e.g. humans and other living organisms, and non-living items like buildings), together with the associated pathways and processes potentially leading to negative effects on human health and the environment. The aquatic environment may contain thousands of compounds, many of them still unknown, that can pose a risk to ecosystems and human health. Due to the unquestionable importance of the aquatic environment, one of the main challenges in the field of exposure science is the comprehensive characterization and evaluation of complex environmental mixtures beyond the classical/priority contaminants to new emerging contaminants. The role of advanced analytical chemistry to identify and quantify potential chemical risks, that might cause adverse effects to the aquatic environment, is essential. In this paper, we present the strategies and tools that analytical chemistry has nowadays, focused on chromatography hyphenated to (high-resolution) mass spectrometry because of its relevance in this field. Key issues, such as the application of effect direct analysis to reduce the complexity of the sample, the investigation of the huge number of transformation/degradation products that may be present in the aquatic environment, the analysis of urban wastewater as a source of valuable information on our lifestyle and substances we consumed and/or are exposed to, or the monitoring of drinking water, are discussed in this article. The trends and perspectives for the next few years are also highlighted, when it is expected that new developments and tools will allow a better knowledge of chemical composition in the aquatic environment. This will help regulatory authorities to protect water bodies and to advance towards improved regulations that enable practical and efficient abatements for environmental and public health protection

Effect-Directed Analysis of Municipal Landfill Soil Reveals Novel Developmental Toxicants in the Zebrafish Danio rerio

Effect-directed analysis (EDA) is an approach used to identify (unknown) contaminants in complex samples which cause toxicity, using a combination of biology and chemistry. The goal of this work was to apply EDA to identify developmental toxicants in soil samples collected from a former municipal landfill site. Soil samples were extracted, fractionated, and tested for developmental effects with an embryotoxicity assay in the zebrafish Danio rerio. Gas chromatograph mass selective detection (GC-MSD) chemical screening was used to reveal candidate developmental toxicants in fractions showing effects. In a parallel study, liquid chromatography-hybrid linear ion trap Orbitrap mass spectrometry was also applied to one polar subfraction (Hoogenboom et al. J. Chromatogr. A2009, 1216, 510-519). EDA resulted in the identification of a number of previously unknown developmental toxicants, which were confirmed to be present in soil by GC-MS. These included 11H-benzo[b]fluorene, 9-methylacridine, 4-azapyrene, and 2-phenylquinoline, as well as one known developmental toxicant (retene). This work revealed the presence of novel contaminants in the environment that may affect vertebrate development, which are not subject to monitoring or regulation under current soil quality assessment guidelines. © 2011 American Chemical Society

Identification strategy for unknown pollutants using high-resolution mass spectrometry: Androgen-disrupting compounds identified through effect-directed analysis

Effect-directed analysis has been applied to a river sediment sample of concern to identify the compounds responsible for the observed effects in an in vitro (anti-)androgenicity assay. For identification after non-target analysis performed on a high-resolution LTQ-Orbitrap, we developed a de novo identification strategy including physico-chemical parameters derived from the effect-directed analysis approach. With this identification strategy, we were able to handle the immense amount of data produced by non-target accurate mass analysis. The effect-directed analysis approach, together with the identification strategy, led to the successful identification of eight androgen-disrupting compounds belonging to very diverse compound classes: an oxygenated polyaromatic hydrocarbon, organophosphates, musks, and steroids. This is one of the first studies in the field of environmental analysis dealing with the difficult task of handling the large amount of data produced from non-target analysis. The combination of bioassay activity assessment, accurate mass measurement, and the identification and confirmation strategy is a promising approach for future identification of environmental key toxicants that are not included as priority pollutants in monitoring programs

Masking effect of anti-androgens on androgenic activity in European river sediment unveiled by effect-directed analysis

This study shows that the androgen receptor agonistic potency is clearly concealed by the effects of androgen receptor antagonists in a total sediment extract, demonstrating that toxicity screening of total extracts is not enough to evaluate the full in vitro endocrine disrupting potential of a complex chemical mixture, as encountered in the environment. The anti-androgenic compounds were masking the activity of androgenic compounds in the extract with relatively high anti-androgenic potency, equivalent to 200 nmol flutamide equivalents/g dry weight. A two-step serial liquid chromatography fractionation of the extract successfully separated anti-androgenic compounds from androgenic compounds, resulting in a total androgenic potency of 3,820 pmol dihydrotestosterone equivalents/g dry weight. The fractionation simplified the chemical identification analysis of the original complex sample matrix. Seventeen chemical structures were tentatively identified. Polyaromatic hydrocarbons, a technical mixture of nonylphenol and dibutyl phthalate were identified to contribute to the anti-androgenic potency observed in the river sediment sample. With the GC/MS screening method applied here, no compounds with AR agonistic disrupting potencies could be identified. Seventy-one unidentified peaks, which represent potentially new endocrine disrupters, have been added to a database for future investigation

Identification of major dioxin-like compounds and androgen receptor antagonist in acid-treated tissue extracts of high trophic-level animals

We evaluated the applicability of combining in vitro bioassays with instrument analyses to identify potential endocrine disrupting pollutants in sulfuric acid-treated extracts of liver and/or blubber of high trophic-level animals. Dioxin-like and androgen receptor (AR) antagonistic activities were observed in Baikal seals, common cormorants, raccoon dogs, and finless porpoises by using a panel of rat and human cell-based chemical-activated luciferase gene expression (CALUX) reporter gene bioassays. On the other hand, no activity was detected in estrogen receptor α (ERα)-, glucocorticoid receptor (GR)-, progesterone receptor (PR)-, and peroxisome proliferator-activated receptor γ2 (PPARγ2)-CALUX assays with the sample amount applied. All individual samples (n = 66) showed dioxin-like activity, with values ranging from 21 to 5500 pg CALUX-2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (TEQ)/g-lipid. Because dioxins are expected to be strong contributors to CALUX-TEQs, the median theoretical contribution of dioxins calculated from the result of chemical analysis to the experimental CALUX-TEQs was estimated to explain up to 130% for all the tested samples (n = 54). Baikal seal extracts (n = 31), but not other extracts, induced AR antagonistic activities that were 8-150 μg CALUX-flutamide equivalent (FluEQ)/g-lipid. p,p′-DDE was identified as an important causative compound for the activity, and its median theoretical contribution to the experimental CALUX-FluEQs was 59% for the tested Baikal seal tissues (n = 25). Our results demonstrate that combining in vitro CALUX assays with instrument analysis is useful for identifying persistent organic pollutant-like compounds in the tissue of wild animals on the basis of in vitro endocrine disruption toxicity. © 2011 American Chemical Society

The NORMAN Association and the European Partnership for Chemicals Risk Assessment (PARC): let’s cooperate! [Commentary]

The Partnership for Chemicals Risk Assessment (PARC) is currently under development as a joint research and innovation programme to strengthen the scientific basis for chemical risk assessment in the EU. The plan is to bring chemical risk assessors and managers together with scientists to accelerate method development and the production of necessary data and knowledge, and to facilitate the transition to next-generation evidence-based risk assessment, a non-toxic environment and the European Green Deal. The NORMAN Network is an independent, well-established and competent network of more than 80 organisations in the field of emerging substances and has enormous potential to contribute to the implementation of the PARC partnership. NORMAN stands ready to provide expert advice to PARC, drawing on its long experience in the development, harmonisation and testing of advanced tools in relation to chemicals of emerging concern and in support of a European Early Warning System to unravel the risks of contaminants of emerging concern (CECs) and close the gap between research and innovation and regulatory processes. In this commentary we highlight the tools developed by NORMAN that we consider most relevant to supporting the PARC initiative: (i) joint data space and cutting-edge research tools for risk assessment of contaminants of emerging concern; (ii) collaborative European framework to improve data quality and comparability; (iii) advanced data analysis tools for a European early warning system and (iv) support to national and European chemical risk assessment thanks to harnessing, combining and sharing evidence and expertise on CECs. By combining the extensive knowledge and experience of the NORMAN network with the financial and policy-related strengths of the PARC initiative, a large step towards the goal of a non-toxic environment can be taken

The NORMAN Suspect List Exchange (NORMAN-SLE): facilitating European and worldwide collaboration on suspect screening in high resolution mass spectrometry

Background: The NORMAN Association (https://www.norman-.network.com/) initiated the NORMAN Suspect List Exchange (NORMAN-SLE; https://www.norman-.network.com/nds/SLE/) in 2015, following the NORMAN collaborative trial on non-target screening of environmental water samples by mass spectrometry. Since then, this exchange of information on chemicals that are expected to occur in the environment, along with the accompanying expert knowledge and references, has become a valuable knowledge base for "suspect screening" lists. The NORMAN-SLE now serves as a FAIR (Findable, Accessible, Interoperable, Reusable) chemical information resource worldwide.Results: The NORMAN-SLE contains 99 separate suspect list collections (as of May 2022) from over 70 contributors around the world, totalling over 100,000 unique substances. The substance classes include per- and polyfluoroalkyl substances (PFAS), pharmaceuticals, pesticides, natural toxins, high production volume substances covered under the European REACH regulation (EC: 1272/2008), priority contaminants of emerging concern (CECs) and regulatory lists from NORMAN partners. Several lists focus on transformation products (TPs) and complex features detected in the environment with various levels of provenance and structural information. Each list is available for separate download. The merged, curated collection is also available as the NORMAN Substance Database (NORMAN SusDat). Both the NORMAN-SLE and NORMAN SusDat are integrated within the NORMAN Database System (NDS). The individual NORMAN-SLE lists receive digital object identifiers (DOIs) and traceable versioning via a Zenodo community (https:// zenodo.org/communities/norman-.sle), with a total of > 40,000 unique views, > 50,000 unique downloads and 40 citations (May 2022). NORMAN-SLE content is progressively integrated into large open chemical databases such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the US EPA's CompTox Chemicals Dashboard (https://comptox. epa.gov/dashboard/), enabling further access to these lists, along with the additional functionality and calculated properties these resources offer. PubChem has also integrated significant annotation content from the NORMAN-SLE, including a classification browser (https://pubchem.ncbi.nlm.nih.gov/classification/#hid=101).Conclusions: The NORMAN-SLE offers a specialized service for hosting suspect screening lists of relevance for the environmental community in an open, FAIR manner that allows integration with other major chemical resources. These efforts foster the exchange of information between scientists and regulators, supporting the paradigm shift to the "one substance, one assessment" approach. New submissions are welcome via the contacts provided on the NORMAN-SLE website (https://www.norman-.network.com/nds/SLE/)