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A variety of anthropogenic compounds has been found to be capable of disrupting the endocrine systems of organisms, in laboratory studies as well as in wildlife. The most widely described endpoint is estrogenicity, but other hormonal disturbances, e.g., thyroid hormone disruption, are gaining more and more attention. Here, we present a review and chemical characterization, using principal component analysis, of organic compounds that have been tested for their capacity to bind competitively to the thyroid hormone transport protein transthyretin (TTR). The database contains 250 individual compounds and technical mixtures, of which 144 compounds are defined as TTR binders. Almost one third of these compounds (n = 52) were even more potent than the natural hormone thyroxine (T₄). The database was used as a tool to assist in the identification of thyroid hormone-disrupting compounds (THDCs) in an effect-directed analysis (EDA) study of a sediment sample. Two compounds could be confirmed to contribute to the detected TTR-binding potency in the sediment sample, i.e., triclosan and nonylphenol technical mixture. They constituted less than 1 % of the TTR-binding potency of the unfractionated extract. The low rate of explained activity may be attributed to the challenges related to identification of unknown contaminants in combination with the limited knowledge about THDCs in general. This study demonstrates the need for databases containing compound-specific toxicological properties. In the framework of EDA, such a database could be used to assist in the identification and confirmation of causative compounds focusing on thyroid hormone disruption.

Marine organisms produce an array of biologically active natural products, many of which have unique structures that have not been found in terrestrial organisms. Hence, marine algae provide a unique source of bioactive compounds. The present study investigated 19 marine algae and one seagrass collected from Torquay beach, Victoria, Australia. High-performance thin-layer chromatography (HPTLC) hyphenated with microchemical (DPPH•, p-anisaldehyde, and Fast Blue B) and biochemical (α-amylase and acetylcholine esterase (AChE) enzymatic) derivatizations was used to evaluate antioxidant activity, presence of phytosterols and phenolic lipids, α-amylase and AChE inhibitory activities of extract components. Significant α-amylase and AChE inhibitory activities were observed in samples 2, 6, 8 and 10. Antioxidant activities in the samples were found to be correlated to phytosterol content (R2 = 0.78), but was not found to be related to either α-amylase or AChE inhibitory activities. α-Amylase inhibitory activities were correlated to AChE inhibition (R2 = 0.77) and attributed to the phytosterol content, based on the similar peak position in the chromatograms with the β-sitosterol chromatogram. Samples 1, 8, and especially sample 20, were found to contain phenolic lipids (alkyl resorcinol derivatives) with significant antioxidant activities. The results suggest that these marine species have a significant number of bioactive compounds that warrant further investigation.

Acetylcholinesterase (AChE) inhibitors are an important class of neuroactive chemicals that are often detected in aquatic and terrestrial environments. The correct functionality of the AChE enzyme is linked to many important physiological processes such as locomotion and respiration. Consequently, it is necessary to develop new analytical strategies to identify harmful AChE inhibitors in the environment. It has been shown that mixture effects and oxidative stress may jeopardize the application of in vivo assays for the identification of AChE inhibitors in the environment. To confirm that in vivo AChE assays can be successfully applied when dealing with complex mixtures, an extract from river water impacted by non-treated wastewater was bio-tested using the acute toxicity fish embryo test (FET) and AChE inhibition assay with zebrafish. The zebrafish FET showed high sensitivity for the extract (LC10 = relative extraction factor 2.8) and we observed a significant inhibition of the AChE (40%, p  < 0.01) after 4-day exposure. Furthermore, the extract was chromatographically fractionated into a total of 26 fractions to dilute the mixture effect and separate compounds according to their physico-chemical properties. As expected, non-specific acute effects (i.e., mortality) disappeared or evenly spread among the fractions, while AChE inhibition was still detected in five fractions. Chemical analysis did not detect any known AChE inhibitors in these active fractions. These results confirm that the AChE assay with Danio rerio can be applied for the detection of neuroactive effects induced in complex environmental samples, but also, they highlight the need to increase analytical and identification techniques for the detection of neurotoxic substances.

Effect-directed analysis (EDA) that combines effect-based methods (EBMs) with high-performance thin-layer chromatography (HPTLC) is a useful technique for spatial, temporal, and process-related effect evaluation and may provide a link between effect testing and responsible substance identification. In this study, a yeast multi endocrine-effect screen (YMEES) for the detection of endocrine effects is combined with HPTLC. Simultaneous detection of estrogenic, androgenic, and gestagenic effects on the HPTLC plate is achieved by mixing different genetically modified Arxula adeninivorans yeast strains, which contain either the human estrogen, androgen, or progesterone receptor. Depending on the yeast strain, different fluorescent proteins are formed when an appropriate substance binds to the specific hormone receptor. This allows to measure hormonal effects at different wavelengths. Two yeast cell application approaches, immersion and spraying, are compared. The sensitivity and reproducibility of the method are shown by dose-response investigations for reference compounds. The spraying approach indicated similar sensitivities and higher precisions for the tested hormones compared to immersion. The EC10s for estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 5α-dihydrotestosterone (DHT), and progesterone (P4) were 95, 1.4, 10, 7.4, and 15 pg/spot, respectively. Recovery rates of E1, E2, EE2, DHT, and P4 between 88 and 120% show the usability of the general method in combination with sample enrichment by solid phase extraction (SPE). The simultaneous detection of estrogenic, androgenic, and gestagenic effects in wastewater and surface water samples demonstrates the successful application of the YMEES in such matrices. This promising method allows us to identify more than one endocrine effect on the same HPTLC plate, which saves time and material. The method could be used for comparison, evaluation, and monitoring of different river sites and wastewater treatment steps and should be tested in further studies.

The main goal of this study was to perform an ecotoxicological profiling of terrestrial and aquatic cyanobacterial strains found in different soils or in toxic cyanobacterial blooms in Vojvodina region, Serbia, using the effect-directed analysis (EDA) approach. The applied procedure was based on a series of in vitro or small-scale bioassays covering multiple endpoints in combination with advanced chemical analytical protocols. Non-selective and non-target preparation techniques were used for the extraction of a broad range of chemical compounds present in three terrestrial (Anabaena Č2, Anabaena Č5, Nostoc S8) and three aquatic (Nostoc Z1, Phormidium Z2, Oscillatoria K3) strains. Ecotoxicological endpoints addressed included evaluation of the fish cytotoxicity in vitro (acute toxicity), algal growth inhibition (chronic toxicity), and interaction with cellular detoxification mechanisms. All cyanobacterial strains tested in the 1st tier EDA showed significant effects in terms of chronic toxicity and interaction with cellular detoxification. Three major fractions of different polarities were further tested in the 2nd tier, using bioassays which showed the strongest response: induction of CYP1A1 biotransformation enzyme and inhibition of zebrafish organic anion (Oatp1d1) and cation (Oct1) uptake transporters. Oscillatoria K3 strain was selected for a more detailed 3rd tier EDA, and the obtained results revealed that positive sub-fractions possess polar anion and cation compounds that are reactive to both uptake transporters, and compounds responsible for the strongest effects have a pronounced lipophilic character. Apart from lipophilic non-polar compounds that represent typical phase I substrates, sub-fractions that contained polar substances are also shown to significantly induce CYP1A1.

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.

The stem bark of black locust (Robinia pseudoacacia L.) was extracted, and nine antioxidant compounds (R1–R9) were detected by high-performance thin-layer chromatography combined with the radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assay, multi-detection, and heated electrospray high-resolution mass spectrometry. For structure elucidation, the methanolic crude extract was fractionated by solid-phase extraction, and the compounds were isolated by reversed-phase high-performance liquid chromatography with diode array detection. The structures of isolated compounds were elucidated by nuclear magnetic resonance and attenuated total reflectance Fourier-transform infrared spectroscopy as well as gas chromatography-mass spectrometry to determine the double bond position. 3-O-Caffeoyl oleanolic acid (R1), oleyl (R2), octadecyl (R3), gadoleyl (R4), eicosanyl (R5), (Z)-9-docosenyl (R6), docosyl (R7), tetracosyl (R8), and hexacosanyl (R9) caffeates were identified. While R1 has been reported in R. pseudoacacia stem bark, the known R3, R5, R7, R8, and R9 are described for the first time in this species, and the R2, R4, and R6 are new natural compounds. All nine caffeates demonstrated antioxidant activity. The antioxidant effects of the isolated compounds R1–R8 were quantified by a microplate DPPH• assay, with values ranging from 0.29 to 1.20 mol of caffeic acid equivalents per mole of isolate.

Introduction Endocrine disrupting chemicals (EDCs) are present in the environment and can have serious effects on humans and wildlife. For the establishment of environmental quality guidelines and regulation of EDCs, a better understanding and knowledge of the occurrence and the behavior of environmental EDCs is necessary. The aim of the present study was to comprehensively identify substances that are responsible for the estrogenic effect of an environmental sediment sample taken from the river Elbe/Germany. Discussion The estrogenic effect of the organic sediment extract was determined using the yeast–estrogen–screen (YES). The sample was fractionated by liquid chromatography (LC) for effect directed analysis. The composition of estrogen-active fractions was further investigated by gas chromatography–mass spectrometry and high-resolution LC–MS analysis. The composition of the environmental sample was rebuilt with pure compounds in order to assess the partition of estrogenic activity caused by the identified compounds. The organic sediment extract showed an estrogenic potential of 1.9 ± 0.4 ng/g ethinylestradiol equivalents in the sediment. The most prominent contaminants with an estrogenic potential were 17β-estradiol, estrone, and 4-iso-nonylphenols, but other xenoestrogens like bisphenol A and stigmasterol could be found as well. A rebuild of the sample was measured in the YES in order to investigate mixture effects. About 67 % of the observed estrogenic effect in the sediment extract could be explained by a mixture which contained all identified compounds. Chlorophene ( o -benzyl- p -chlorophenol)—a widely used antiseptic that was also identified in the sediment extract—has xenoestrogenic properties in the YES that are in the range of other xenoestrogens like 4- n -nonylphenol. This is the first report on chlorophene acting as a xenoestrogen.

This study investigates the pancreatic lipase inhibitory activity of oleogum resins from various Boswellia species, integrating high-performance thin-layer chromatography (HPTLC)‒effect-directed analysis (EDA) with molecular docking to evaluate bioactive compounds. Pancreatic lipase, a key enzyme in dietary fat digestion, is a crucial target in anti-obesity therapies. HPTLC‒EDA revealed that 3-acetyl-11-keto-β-boswellic acid (AKBA), β-boswellic acid (BA), and 3-acetyl-β-boswellic acid (ABA) exhibited notable inhibitory activities, indicated by distinct inhibition zones on HPTLC bioautograms. Among the species tested, Boswellia serrata demonstrated the strongest inhibitory potential, while Boswellia frereana and Boswellia neglecta showed limited activity. Molecular docking studies supported these findings by elucidating the binding interactions of AKBA, BA, and ABA with the pancreatic lipase (PDB ID: 1ETH). The hydrogen bond(s) with Lys81 and/or Arg112 were found to be responsible for the inhibitory effects of AKBA, BA, and ABA on the pancreatic lipase. Unlike orlistat, AKBA, BA, and ABA could not establish hydrogen bonds with three pivotal residues (Ser153, Gly77, and His152), and therefore their lower inhibitory effects may be related to the lack of these binding interactions. These findings suggest that, while Boswellia -derived compounds show promise as natural lipase inhibitors, structural optimization may be required to enhance their potency.

Extensive monitoring programs on chemical contamination are run in many European river basins. With respect to the implementation of the European Union (EU) Water Framework Directive (WFD), these programs are increasingly accompanied by monitoring the ecological status of the river basins. Assuming an impact of chemical contamination on the ecological status, the assignment of effects in aquatic ecosystems to those stressors that cause the effects is a prerequisite for taking political or technical measures to achieve the goals of the WFD. Thus, one focus of present European research is on toxicant identification in European river basins in order to allow for a reduction of toxic pressure on aquatic ecosystems according to the WFD. An overview is presented on studies that were performed to link chemical pollution in European river basins to measurable ecotoxic effects. This includes correlation-based approaches as well as investigations that apply effect-directed analysis (EDA) integrating toxicity testing, fractionation and non-target chemical analysis. Effect-based key toxicants that were identified in European surface waters are compiled and compared to EU priority pollutants. Further needs for research are identified. Studies on the identification of effect-based key toxicants focused on mutagenicity, aryl hydrocarbon receptor-mediated effects, endocrine disruption, green algae, and invertebrates. The identified pollutants include priority pollutants and other well-known environmental pollutants such as polycyclic aromatic hydrocarbons, polychlorinated dibenzo-p-dioxins, furans, and biphenyls, nonylphenol, some pesticides and tributyltin, but also other compounds that were neither considered as environmental pollutants before nor regulated such as substituted phenols, natural or synthetic estrogens and androgens, dinaphthofurans, 2-(2-naphthalenyl)benzothiophene, and N-phenyl-2-naphthylamine. Individual studies at specific sites in a European river basin demonstrated the power of combined biological and chemical analytical approaches and, particularly, of effect-directed analysis. However, the available information on effect-based key toxicants is very limited with respect to the entirety of rivers possibly at risk due to chemical contamination and with respect to toxicological endpoints considered at a specific site. A relatively broad basis of information exists only for estrogenicity and aryl hydrocarbon Ah-receptor-mediated effects. The development of tools and strategies for an identification of key toxicants on a broader scale are a challenging task for the next years. Since investigations dealing with toxicant identification are too labor and cost-intensive for monitoring purposes, they have to be focused on the key sites in a river basin. These should include hot spots of contamination, particularly if there is evidence that they might pose a risk for downstream areas, but may also involve accumulation zones in the lower reach of a river in order to get an integrated picture on the contamination of the basin. Recommendations and Perspectives. While EDA is almost exclusively based on measurable effects in in vitro and in vivo biotests to date, an increasing focus in the future should be on the integration of EDA into Ecological Risk Assessment and on the development of tools to confirm EDA-determined key toxicants as stressors in populations, communities and ecosystems. Considering these requirements and applied in a focused way, toxicant identification may significantly help to implement the Water Framework Directive by providing evidence on the main stressors and possible mitigation measures in order to improve the ecological status of a river ecosystem.