An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice.

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines.

Are changes in vitellogenin concentrations in fish reliable indicators of chemical-induced endocrine activity?

Vitellogenin (VTG), a biomarker for endocrine activity, is a mechanistic component of the regulatory assessment of potential endocrine-disrupting properties of chemicals. This review of VTG data is based on changes reported for 106 substances in standard fish species. High intra-study and inter-laboratory variability in VTG concentrations was confirmed, as well as discrepancies in interpretation of results based on large differences between fish in the dilution water versus solvent control, or due to the presence of outlier measurements. VTG responses in fish were ranked against predictions for estrogen receptor agonist activity and aromatase inhibition from bioactivity model output and ToxCast in vitro assay results, respectively. These endocrine mechanisms explained most of the VTG responses in the absence of systemic toxicity, the magnitude of the VTG response being proportional to the in vitro potency. Interpretation of the VTG data was sometimes confounded by an alternative endocrine mechanism of action. There was evidence for both false positive and negative responses for VTG synthesis, but overall, it was rare for substances without endocrine activity in vitro to cause a concentration-dependent VTG response in fish in the absence of systemic toxicity. To increase confidence in the VTG results, we recommend improvements in the VTG measurement methodologies and greater transparency in reporting of VTG data (including quality control criteria for assay performance). This review supports the application of New Approach Methodologies (NAMs) by demonstrating that endocrine activity in vitro from mammalian cell lines is predictive for in vivo VTG response in fish, suggesting that in vitro mechanistic data could be used more broadly in decision-making to help reduce animal testing.

TWAc-Check: A New Approach to Determine the Appropriate Use of Time-Weighted Average Concentration in Aquatic Risk Assessment.

In pesticide risk assessment, regulatory acceptable concentrations for surface water bodies (RACsw,ch) are used that are derived from standard studies with continuous exposure of organisms to a test compound for days or months. These RACsw,ch are compared with the maximum tested concentration of more realistic exposure scenarios. However, the actual exposure duration could be notably shorter (e.g., hours) than the standard study, which intentionally leads to an overly conservative Tier 1 risk assessment. This discrepancy can be addressed in a risk assessment using the time-weighted average concentration (TWAc). In Europe, the applicability of TWAc for a particular risk assessment is evaluated using a complex decision scheme, which has been controversial; thus we propose an alternative approach: We used TWAc-check (which is based on the idea that the TWAc concept is just a model for aquatic risk assessment) to test whether the use of a TWAc is appropriate for such assessment. The TWAc-check method works by using predicted-measured diagrams to test how well the TWAc model predicts experimental data from peak exposure experiments. Overestimated effects are accepted because the conservatism of the TWAc model is prioritized over the goodness of fit. We illustrate the applicability of TWAc-check by applying it to various data sets for different species and substances. We demonstrate that the applicability is case dependent. Specifically, TWAc-check correctly identifies that the use of TWAc is not appropriate for early onset of effects or delayed effects. The proposed concept shows that the time window is a decisive factor as to whether or not the model is acceptable and that this concept can be used as a potential refinement option prior to the use of toxicokinetic-toxicodynamic models. Environ Toxicol Chem 2022;41:1778-1787. © 2022 Bayer AG. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Extensive rain events have a more substantial impact than advanced effluent treatment on the endocrine-disrupting activity in an effluent-dominated small river.

Wastewater treatment plants (WWTPs) remain an important primary source of emission for endocrine-disrupting compounds in the environment. As an advanced wastewater treatment process, ozonation is known to reduce endocrine-disrupting activity. However, it remains unclear to which extend improved wastewater treatment may reduce the endocrine-disrupting activity in the receiving water body. The present study investigated possible factors for the endocrine-disrupting activity in a small receiving water body, the Wurm River (North-Rhine Westphalia, Germany), up- and downstream of a local WWTP. The cell-based reporter gene CALUX® assay was applied to identify the endocrine-disrupting activity in the water, sediment, and suspended particulate matter. The water phase and the effluent sampling were primarily driven by applying the full-scale effluent ozonation (sampling campaigns in June 2017 and March 2019). In contrast, the sediment sampling aimed to compare the particle-bound endocrine-disrupting activity during dry (June 2017) and rainy summer (June 2018) seasons. The water phase showed low to moderate estrogenic/antiandrogenic activity. Advanced effluent treatment by ozonation led to a complete reduction of the endocrine-disrupting activity according to the limit of detection of the CALUX® assays. The suspended particulate matter originated from the water phase of the second sampling campaign revealed antiandrogenic activity only. Sediments at the sampling sites along the local WWTP revealed higher estrogenic and antiandrogenic activity after extensive rain events and were not affected by the ozonated effluent. Fluctuation patterns of the endocrine-disrupting activity in sediments were in line with fluctuated concentrations of polycyclic aromatic hydrocarbons. Rainwater overflow basin release was suggested as a vector for particle-bound and dissolved endocrine-disrupting activity in the receiving water body. The present study underlined the necessity for monitoring both water and sediment phases to achieve reliable profiling of the endocrine-disrupting activity. The receptor-mediated CALUX® assays were proven to be suitable for investigating the endocrine-disrupting activity distribution in different river compartments and WWTP effluents.

Improvement of wastewater and water quality via a full-scale ozonation plant? - A comprehensive analysis of the endocrine potential using effect-based methods.

Micropollutants (MPs), especially endocrine disrupting compounds (EDCs), are mainly released from WWTPs into surface water bodies and can subsequently lead to adverse effects in biota. Treatment with ozone proved to be a suitable method for eliminating such MPs. This method was implemented at the WWTP Aachen-Soers by commissioning the largest full-scale ozonation plant in Europe at the moment. Recently, effect-based methods (EBMs) have been successfully proved for compliance monitoring, e.g. estrogenic compounds. Therefore, the impact of ozone treatment on endocrine potential (agonistic and antagonistic) of treated wastewater was investigated using the ERα- and AR CALUX assays. Additionally, the impact on the receiving stream and a potential preload of the water body was assessed. Therefore, the current study could deal as a case study for small rivers being highly impacted by WWTPs. The estrogenic potential was nearly fully eliminated after ozone treatment. Contrary, the antagonistic (anti-estrogenic and anti-androgenic) potential did not show a clear elimination pattern after ozone treatment independent of the applied ozone dosage and control system. Therefore, further investigations are required regarding the antagonistic potential. Additionally, preloading of the receiving stream was found during the study period. One significant impact is a rain overflow basin (ROB) located upstream of the WWTP effluent. The highest endocrine potential was found after a ROB overflow (2.7 ng EEQ/L, 2.4 µg TMX-EQ/L, 104 µg FLU-EQ/L), suggesting that such runoff events after a heavy rainfall may act as a driver of endocrine loading to the water body. This manuscript contributes significantly to the basic understanding of the efficiency of eliminating the endocrine potential of ozone treatment by, e.g., showing that there is a further need for improving the removal efficiency of antagonistic potential. Moreover, it highlights the need to include other point sources, such as ROBs, to assess polluted surface waters comprehensively.

Assessing the genotoxic potential of freshwater sediments after extensive rain events - Lessons learned from a case study in an effluent-dominated river in Germany.

Wastewater treatment plant effluents and releases from rainwater overflow basins can contribute to the input of genotoxic micropollutants in aquatic ecosystems. Predominantly lipophilic genotoxic compounds tend to sorb to particulate matter, making sediment a source and a sink of pollution. Therefore, the present study aims to investigate the genotoxic potential of freshwater sediments (i) during the dry period and (ii) after extensive rain events by collecting sediment samples in one small anthropogenically impacted river in Germany up- and downstream of the local wastewater treatment plant. The Micronucleus and Ames fluctuation assays with Salmonella typhimurium strains TA98, TA100, YG1041, and YG1042 were used to assess the genotoxic potential of organic sediment extracts. For evaluation of possible genotoxicity drivers, target analysis for 168 chemical compounds was performed. No clastogenic effects were observed, while the genotoxic potential was observed at all sampling sites primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. Freshwater sediments' genotoxic potential increased after extensive rain events due to sediment perturbation and the rainwater overflow basin release. In the present study, the rainwater overflow basin was a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of the bacterial Salmonella typhimurium strains YG1041 and YG1042 to organic sediment extracts to assess the different classes of genotoxic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.

Synthesis of biologically active peptide nucleic acid-peptide conjugates by sortase-mediated ligation.

Sortase A is a transpeptidase that cleaves at a pentapeptide-motif and subsequently transfers the acyl component to a nucleophile containing N-terminal oligoglycines. We investigate the reaction conditions of the sortase-mediated ligation and demonstrate a useful application by the synthesis of a peptide nucleic acid-cell-penetrating peptide chimera, the reaction equilibrium of which can be shifted in favor of the product by dialyzing out the low molecular weight byproduct. The synthesized conjugate exhibits dose-dependent antisense activity.

Structural requirements for cellular uptake and antisense activity of peptide nucleic acids conjugated with various peptides.

Peptide nucleic acids (PNAs) have shown great promise as potential antisense drugs; however, poor cellular delivery limits their applications. Improved delivery into mammalian cells and enhanced biological activity of PNAs have been achieved by coupling to cell-penetrating peptides (CPPs). Structural requirements for the shuttling ability of these peptides as well as structural properties of the conjugates such as the linker type and peptide position remained controversial, so far. In the present study an 18mer PNA targeted to the cryptic splice site of a mutated beta-globin intron 2, which had been inserted into a luciferase reporter gene coding sequence, was coupled to various peptides. As the peptide lead we used the cell-penetrating alpha-helical amphipathic peptide KLAL KLAL KAL KAAL KLA-NH2 [model amphipathic peptide (MAP)] which was varied with respect to charge and structure-forming properties. Furthermore, the linkage and the localization of the attached peptide (C- vs N-terminal) were modified. Positive charge as well as helicity and amphipathicity of the KLA peptide was all required for efficient dose-dependent correction of aberrant splicing. The highest antisense effect was reached within 4 h without any transfection agent. Stably linked conjugates were also efficient in correction of aberrant splicing, suggesting that a cleavable disulfide bond between CPP and PNA is clearly not essential. Moreover, the placement of the attached peptide turned out to be crucial for attaining antisense activity. Coadministration of endosome disrupting agents such as chloroquine or Ca2+ significantly increased the splicing correction efficiency of some conjugates, indicating the predominant portion to be sequestered in vesicular compartments.

Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.

In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.