Effect-directed analysis (EDA) has shown its added value for the detection and identification of compounds with varying toxicological properties in water quality research. However, for routine toxicity assessment of multiple toxicological endpoints, current EDA is considered labor intensive and time consuming. To achieve faster EDA and identification, a high-throughput (HT) EDA platform, coupling a downscaled luminescent Ames and cell-based reporter gene assays with a high-resolution fraction collector and UPLC-QTOF MS, was developed. The applicability of the HT-EDA platform in the analysis of aquatic samples was demonstrated by analysis of extracts from WWTP influent, effluent and surface water. Downscaled assays allowed detection of mutagenicity and androgen, estrogen and glucocorticoid agonism following high-resolution fractionation in 228 fractions. From 8 masses tentatively identified through non-target analysis, 2 masses were further investigated and chemically and biologically confirmed as the mutagen 1,2,3-benzotriazole and the androgen androstenedione. The compatibility of the high-throughput EDA platform with analysis of water samples and the incorporation of mutagenic and endocrine disruption endpoints allow for future application in routine monitoring in drinking water quality control and improved identification of (emerging) mutagens and endocrine disruptors.
The AR-EcoScreen is a widely used reporter assay for the detection of androgens and anti-androgens. Endogenous expression of glucocorticoid receptors and their affinity for the androgen responsive element that drives reporter expression, however, makes the reporter cells sensitive to interference by glucocorticoids and less specific for (anti-)androgens. To create a glucocorticoid insensitive derivative of the AR-EcoScreen, CRISPR/Cas9 genome editing was used to develop glucocorticoid receptor knockout mutants by targeting various sites in the glucocorticoid gene. Two mutant cell lines were further characterized and validated against the unmodified AR-EcoScreen with a set of 19 environmentally relevant chemicals and a series of environmental passive sampler extracts with (anti-)androgenic activity. Sequencing of the targeted sites revealed premature stop codons following frame-shift mutations, leading to an absence of functional glucocorticoid receptor expression. The introduced mutations rendered cell lines insensitive to glucocorticoid activation and caused no significant difference in the responsiveness towards (anti-)androgens, compared to the unmodified AR-EcoScreen cells, allowing the selective, GR-independent, determination of (anti-)androgenicity in environmental passive sampler extracts. The increase in selectivity for (anti-)androgens improves reliability of the AR-EcoScreen and will provide higher accuracy in determining (anti-)androgenic potential when applied in toxicity screening and environmental monitoring of both single compounds and mixtures.
The toxic equivalency concept used for the risk assessment of polychlorinated biphenyls (PCBs) is based on the aryl hydrocarbon receptor (AhR)–mediated toxicity of coplanar dioxin-like (DL) PCBs. Most PCBs in the environment, however, are non-dioxin-like (NDL) PCBs that cannot adopt a coplanar structure required for AhR activation. For NDL-PCBs, no generally accepted risk concept is available because their toxicity is insufficiently characterized. Here, we systematically determined in vitro toxicity profiles for 24 PCBs regarding 10 different mechanisms of action. Prior to testing, NDL-PCB standards were purified to remove traces of DL compounds. All NDL-PCBs antagonized androgen receptor activation and inhibited gap junctional intercellular communication (GJIC). Lower chlorinated NDL-PCBs were weak estrogen receptor (ER) agonists, whereas higher chlorinated NDL-PCBs were weak ER antagonists. Several NDL-PCBs inhibited estradiol-sulfotransferase activity and bound to transthyretin (TTR) but with much weaker potencies than reported for hydroxylated PCB metabolites. AhR-mediated expression of uridine-glucuronyl transferase isozyme UGT1A6 was induced by DL-PCBs only. Hierarchical cluster analysis of the toxicity profiles yielded three separate clusters of NDL-PCBs and a fourth cluster of reference DL-PCBs. Due to small differences in relative potency among congeners, the highly abundant indicator PCBs 28, 52, 101, 118, 138, 153, and 180 also contributed most to the antiandrogenic, (anti)estrogenic, antithyroidal, tumor-promoting, and neurotoxic potencies calculated for PCB mixtures reported in human samples, whereas the most potent AhR-activating DL-PCB-126 contributed at maximum 0.2% to any of these calculated potencies. PCB-168 is recommended as an additional indicator congener, given its relatively high abundance and antiandrogenic, TTR-binding, and GJIC-inhibiting potencies.
In vitro bioassays are valuable tools for screening environmental samples for the presence of bioactive (e.g., endocrine-disrupting) compounds. They can be used to direct chemical analysis of active compounds in toxicity identification and evaluation (TIE) approaches. In the present study, five in vitro bioassays were used to profile toxic potencies in sediments, with emphasis on endocrine disruption. Nonpolar total and acid-treated stable extracts of sediments from 15 locations in the Rhine Meuse estuary area in The Netherlands were assessed. Dioxin-like and estrogenic activities (using dioxin-responsive chemical-activated luciferase gene expression [DR-CALUX] and estrogen-responsive chemical-activated luciferase gene expression [ER-CALUX] assays) as well as genotoxicity (UMU test) and nonspecific toxic potency (Vibrio fischeri assay) were observed in sediment extracts. For the first time, to our knowledge, in vitro displacement of thyroid hormone thyroxine (T4) from the thyroid hormone transport protein thransthyretin by sediment extracts was observed, indicating the presence of compounds potentially able to disrupt T4 plasma transport processes. Antiestrogenic activity was also observed in sediment. The present study showed the occurrence of endocrine-disrupting potencies in sediments from the Dutch delta and the suitability of the ER- and DR-CALUX bioassays to direct endocrine-disruption TIE studies.
The goal of this study is to enhance the identification of unknown compounds in LC chromatograms of untreated raw water used for drinking water production by (1) preconcentration of samples and (2) prioritization of the unknown peaks based on their bioactivity. Preconcentration was done by solid phase extraction (SPE) of 3L large volume (LV) groundwater samples or by 6-week passive sampler (PS) deployment in the groundwater, using divinylbenzene as active phase. Bioactivity of the samples was tested as a decrease in bioluminescence in the Allivibrio fischeri bioassay, which was chosen as a broad-scale bioanalytical tool responding to many different types of pollutants. Samples were collected at three different drinking water production stations with low or high degree of anthropogenic influence. At two stations, different groundwater inlets were sampled. At one station, samples were taken at different stages in the drinking water production process. SPE and PS extracts were used for target analysis and non-target screening, and for testing in the bioassay before and after high-resolution fractionation. More target compounds were detected in the concentrated LV and PS extracts than in a 1 mL direct injection of the water. As expected least compounds and lowest bioassay responses were detected in the raw water from the station with least anthropogenic influence. PS extracts gave much higher bioassay responses than the LV extracts. Both chemical and bioassay analysis of samples collected during subsequent steps in the drinking water production process confirmed the efficient removal of (bioactive) contaminants. In addition to the 4 peaks detected in the bioassay chromatogram of the reference station, the more anthropogenically influenced stations showed additional peaks indicative for the presence of anthropogenic substances. Identification of suspect compounds responsible for these additional peaks will proceed according to a three-step procedure, i.e. based on (1) exact mass and isotopic pattern of compounds available in different suspect lists, (2) fragment ions assigned to a precursor available in spectral libraries, and (3) estimated elemental composition. Reference standards will be obtained for the suspect compounds to confirm their retention time and bioactivity. Results from the identification process are available by the end of May 2020, and will be presented at the SETAC Europe conference.
Humans are exposed to a large number of chemicals from sources such as the environment, food, and consumer products. There is growing concern that human exposure to chemical mixtures, especially during critical periods of development, increases the risk of adverse health effects in newborns or later in life. Historically, the one-chemical-at-a-time approach has been applied both for exposure assessment and hazard characterisation, leading to insufficient knowledge about human health effects caused by exposure to mixtures of chemicals that have the same target. To circumvent this challenge researchers can apply in vitro assays to analyse both exposure to and human health effects of chemical mixtures in biological samples. The advantages of using in vitro assays are: (i) that an integrated effect is measured, taking combined mixture effects into account and (ii) that in vitro assays can reduce complexity in identification of Chemicals of Emerging Concern (CECs) in human tissues. We have reviewed the state-of-the-art on the use of receptor-based in vitro assays to assess human exposure to chemical mixtures and related health impacts. A total of 43 studies were identified, in which endpoints for the arylhydrocarbon receptor (AhR), the estrogen receptor (ER), and the androgen receptor (AR) were used. The majority of studies reported biological activities that could be associated with breast cancer incidence, male reproductive health effects, developmental toxicities, human demographic characteristics or lifestyle factors such as dietary patterns. A few studies used the bioactivities to check the coverage of the chemical analyses of the human samples, whereas in vitro assays have so far not regularly been used for identifying CECs in human samples, but rather in environmental matrices or food packaging materials. A huge field of novel applications using receptor-based in vitro assays for mixture toxicity assessment on human samples and effect-directed analysis (EDA) using high resolution mass spectrometry (HRMS) for identification of toxic compounds waits for exploration. In the future this could lead to a paradigm shift in the way we unravel adverse human health effects caused by chemical mixtures.
This open online textbook on Environmental Toxicology aims at covering the field in its full width, including aspects of environmental chemistry, ecotoxicology, toxicology and risk assessment. With that, it will contribute to improving the quality, continuity and transparency of the education in environmental toxicology. We also want to make sure that fundamental insights on fate and effects of chemicals gained in the past are combined with recent approaches of effect assessment and molecular analysis of mechanisms causing toxicity. The book consists of six chapters, with each chapter being divided into several sub-chapters to enable covering all aspects relevant to the topic. All chapters are designed in a modular way, which each module having clear training goals and being flagged with a number of keywords. Most modules have an average length of 1000-2000 words, a limited number of references, and 3-5 figures and/or tables. A few modules are enlighted with short clips, animations or movies to allow better illustration of the theory. The introduction chapter of the book, for instance, contains a short interview with two key experts reflecting on the development of the field over the past 30 years. The book contains tools for self-study and training, like a (limited) number of questions at the end of each module. For the future we foresee the addition of separate exercises and other tools that may help the student in understanding the theory. The open online textbook was developed by a project team consisting of environmental toxicologists and chemists from six Dutch universities, who drafted the outline of the book and identified authors for each module. Each module is authored by one or two members of the project team. When a topic required expertise not present among the project team, an external expert, often from abroad, was asked to write the module. To guarantee the quality of the book, each module was reviewed by at least one of the members of the project team and also by an international reviewer from outside the project team. The project was supervised by an advisory board, a steering committee and educational advisors. The project team served as the editorial board. The publication of this book on an open online publication platform allows free access to anyone. It is also embedded in learning management systems used in university teaching, thereby giving students easy access. The modular composition of the book will allow teachers to design their “own” book by selecting those modules relevant for the class they teach. This will support flexible use of the book. The publication as an open online book will allow for continuous updating, so it can stay on top of new developments in the field. As it stands, about 100 modules have been finalized, another 30 modules are already available in drafts and currently in the process of being reviewed, and some are still in preparation. In spite of the large number of modules, which do provide a good basis for teaching at the bachelor of science level, we realize the book is still not complete. The book needs more advanced modules that would facilitate teaching at the master of science level and higher, as well as widening the number of topics covered. We would also like to include a broader perspective on regulatory issues so the book covers different geographic regions. Therefore, we will continue working on the book, but we also welcome any suggestions for extending the book. We invite colleagues in environmental toxicology and chemistry to take the initiative to write modules on topics still missing.
PCB 180 is a persistent non-dioxin-like polychlorinated biphenyl (NDL-PCB) abundantly present in food and the environment. Risk characterization of NDL-PCBs is confounded by the presence of highly potent dioxin-like impurities. We used ultrapure PCB 180 to characterize its toxicity profile in a 28-day repeat dose toxicity study in young adult rats extended to cover endocrine and behavioral effects. Using a loading dose/maintenance dose regimen, groups of 5 males and 5 females were given total doses of 0, 3, 10, 30, 100, 300, 1000 or 1700 mg PCB 180/kg body weight by gavage. Dose-responses were analyzed using benchmark dose modeling based on dose and adipose tissue PCB concentrations. Body weight gain was retarded at 1700 mg/kg during loading dosing, but recovered thereafter. The most sensitive endpoint of toxicity that was used for risk characterization was altered open field behavior in females; i.e. increased activity and distance moved in the inner zone of an open field suggesting altered emotional responses to unfamiliar environment and impaired behavioral inhibition. Other dose-dependent changes included decreased serum thyroid hormones with associated histopathological changes, altered tissue retinoid levels, decreased hematocrit and hemoglobin, decreased follicle stimulating hormone and luteinizing hormone levels in males and increased expression of DNA damage markers in liver of females. Dose-dependent hypertrophy of zona fasciculata cells was observed in adrenals suggesting activation of cortex. There were gender differences in sensitivity and toxicity profiles were partly different in males and females. PCB 180 adipose tissue concentrations were clearly above the general human population levels, but close to the levels in highly exposed populations. The results demonstrate a distinct toxicological profile of PCB 180 with lack of dioxin-like properties required for assignment of WHO toxic equivalency factor. However, PCB 180 shares several toxicological targets with dioxin-like compounds emphasizing the potential for interactions.
