Search

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns. In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis. All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium. Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

Objectives. The retinoblastoma (RB) pathway is crucial in the development and progression of many cancers. To better understand the biology of progressive breast cancer (BC), we examined protein expression of the RB pathway in primary BCs and matched axillary lymph node metastases (LM). Methods. Immunohistochemistry was used to evaluate cyclin D1, CDK4/6, RB, phosphorylated RB (pRB), and E2F1 expression in tissue arrays containing cores of 50 primary BCs and matched LM. The number of positive tumor cells and staining intensity were scored. Results. The proteins were localized in the nucleus, while CDK6 was detected in the cytoplasm and CDK4 was found in both. pRB and E2F1 showed higher expression in matched LM than in primary tumors. Expression of these proteins differed significantly by the percentage of positive tumor cells, while proteins in the proximal portion of the RB pathway showed no significant differences. The main path of alteration consisted of high pRB in primary BC, remaining pRB high in the majority of LM, variations occurring in fewer cases. All matched LM of the few primary tumors that had unaltered RB and pRB expression showed changes in RB or pRB expression. Conclusion. Expression of pRB and E2F1 was significantly higher in LM than in primary BC. A majority of cancers with LM showed altered RB or pRB expression, suggesting that proteins downstream in the RB pathway play a critical role in metastatic BC and disease progression. So looking at the RB pathway could be an option for chemotherapy decisions in patients with only few LM.

Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients’ brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling., Graphical abstract, In this article, Pless and colleagues show upregulation of IFNγ signaling in the neurovascular unit of the brain in fatal COVID-19. They show that an hiPSC-derived brain capillary endothelial cell model can be infected with SARS-CoV-2, resulting in similar expression changes, viral replication, and release while endothelial cell integrity is maintained. Infection can be prevented by antibodies or protease inhibitors.

Mixtures extracted from beluga traditional food preparations activated oxidative stress response and the aryl hydrocarbon receptor but did not show any hormone-like effects.

Aims Although soil water deficit is the primary constraint on transpiration globally, the mechanisms by which soil drying and soil properties impact transpiration and stomatal regulation remain elusive. This work aimed to investigate how soil textures and vapor pressure deficit (VPD) impact the relationship between transpiration rate, canopy conductance, and leaf water potential of maize (Zea mays L.) during soil drying. We hypothesize that the decrease in soil–plant hydraulic conductance (Ksp) triggers stomatal closure and the latter is soil specific. Methods Plants were grown in two contrasting soil textures (sand and loam) and exposed to two consecutive VPD levels (1.8 and 2.8 kPa). We measured transpiration rate, canopy conductance, soil and leaf water potentials during soil drying. Results Transpiration rate decreased at higher soil matric potential in sand than in loam at both VPD levels. In sand, high VPD generated a steeper drop in canopy conductance with decreasing leaf water potential. The decrease in canopy conductance was well correlated with the drop in Ksp, which was significantly affected by soil texture. Conclusions Our results demonstrated that variations in canopy conductance were not simply a function of leaf water potential but largely affected by soil hydraulics. These results reinforce a model of stomatal closure driven by a loss in soil hydraulic conductivity. Further studies will determine if soil-specific stomatal regulation exists among species.

The presence of anthropogenic organic micropollutants in rivers poses a long-term threat to surface water quality. To describe and quantify the in-stream fate of single micropollutants, the advection-dispersion-reaction (ADR) equation has been used previously. Understanding the dynamics of the mixture effects and cytotoxicity that are cumulatively caused by micropollutant mixtures along their flow path in rivers requires a new concept. Thus, we extended the ADR equation from single micropollutants to defined mixtures and then to the measured mixture effects of micropollutants extracted from the same river water samples. Effects (single and mixture) are expressed as effect units and toxic units, the inverse of effect concentrations and inhibitory concentrations, respectively, quantified using a panel of in vitro bioassays. We performed a Lagrangian sampling campaign under unsteady flow, collecting river water that was impacted by a wastewater treatment plant (WWTP) effluent. To reduce the computational time, the solution of the ADR equation was expressed by a convolution-based reactive transport approach, which was used to simulate the dynamics of the effects. The dissipation dynamics of the individual micropollutants were reproduced by the deterministic model following first-order kinetics. The dynamics of experimental mixture effects without known compositions were captured by the model ensemble obtained through Bayesian calibration. The highly fluctuating WWTP effluent discharge dominated the temporal patterns of the effect fluxes in the river. Minor inputs likely from surface runoff and pesticide diffusion might contribute to the general effect and cytotoxicity pattern but could not be confirmed by the model-based analysis of the available effect and chemical data.

All chemicals can interfere with cellular membranes and this leads to baseline toxicity, which is the minimal toxicity any chemical elicits. The critical membrane burden is constant for all chemicals; that is, the dosing concentrations to trigger baseline toxicity decrease with increasing hydrophobicity of the chemicals. Quantitative structure-activity relationships, based on hydrophobicity of chemicals, have been established to predict nominal concentrations causing baseline toxicity in human and mammalian cell lines. However, their applicability is limited to hydrophilic neutral compounds. To develop a prediction model that includes more hydrophobic and charged organic chemicals, a mass balance model was applied for mammalian cells (AREc32, AhR-CALUX, PPARγ-BLA, and SH-SY5Y) considering different bioassay conditions. The critical membrane burden for baseline toxicity was converted into nominal concentration causing 10% cytotoxicity by baseline toxicity (IC10,baseline) using a mass balance model whose main chemical input parameter was the liposome-water partition constants (Klip/w) for neutral chemicals or the speciation-corrected Dlip/w(pH 7.4) for ionizable chemicals plus the bioassay-specific protein, lipid, and water contents of cells and media. In these bioassay-specific models, log(1/IC10,baseline) increased with increasing hydrophobicity, and the relationship started to level off at log Dlip/w around 2. The bioassay-specific models were applied to 392 chemicals covering a broad range of hydrophobicity and speciation. Comparing the predicted IC10,baseline and experimental cytotoxicity IC10, known baseline toxicants and many additional chemicals were identified as baseline toxicants, while the others were classified based on specificity of their modes of action in the four cell lines, confirming excess toxicity of some fungicides, antibiotics, and uncouplers. Given the similarity of the bioassay-specific models, we propose a generalized baseline-model for adherent human cell lines: log[1/IC10,baseline (M)] = 1.23 + 4.97 × (1 - e-0.236 log Dlip/w). The derived models for baseline toxicity may serve for specificity analysis in reporter gene and neurotoxicity assays as well as for planning the dosing for cell-based assays.

Suspended particulate matter (SPM) plays an important role in the fate of organic micropollutants in rivers during rain events, when sediments are remobilized and turbid runoff components enter the rivers. Under baseflow conditions, the SPM concentration is low and the contribution of SPM-bound contaminants to the overall risk of organic contaminants in rivers is assumed to be negligible. To challenge this assumption, we explored if SPM may act as a source or sink for all or specific groups of organic chemicals in a small river. The concentrations of over 600 contaminants and the mixture effects stemming from all chemicals in in vitro bioassays were measured for river water, SPM, and the surface sediment after solid-phase extraction or exhaustive solvent extraction. The bioavailable fractions of chemicals and mixture effects were estimated after passive equilibrium sampling of enriched SPM slurries and sediments in the lab. Dissolved compounds dominated the total chemical burden in the water column (water plus SPM) of the river, whereas SPM-bound chemicals contributed up to 46% of the effect burden even if the SPM concentration in rivers was merely 1 mg/L. The equilibrium between water and SPM was still not reached under low-flow conditions with SPM as a source of water contamination. The ratios of SPM-associated to sediment-associated neutral and hydrophobic chemicals as well as the ratios of the mixture effects expressed as bioanalytical equivalent concentrations were close to 1, suggesting that the surface sediment can be used as a proxy for SPM under baseflow conditions when the sampling of a large amount of water to obtain sufficient SPM cannot be realized.

Background Liver metastases are common in patients with breast cancer, and determining the factors associated with such metastases may improve both their early detection and treatment. Given that liver function protein level changes in these patients have not been determined, the aim of our study was to investigate liver function protein level changes over time, spanning 6 months before the detection of liver metastasis to 12 months after. Methods We retrospectively studied 104 patients with hepatic metastasis from breast cancer who were treated at the Departments of Internal Medicine I and the Department of Obstetrics and Gynecology at the Medical University of Vienna between 1980 and 2019. Data were extracted from patient records. Results Aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, lactate dehydrogenase and alkaline phosphatase were significantly elevated when compared to normal range 6 months before the detection of liver metastases (pppp = 0.002) and reduced albumin (p = 0.002) levels at the time of diagnosis were associated with shorter overall survival. Conclusion Liver function protein levels should be considered as potential indicators when screening for liver metastasis in patients with breast cancer. With the new treatment options available, it could lead to prolonged life.

Storm events lead to a mobilization of dissolved and particle-associated organic pollutants that pose a risk to river ecosystems. Target screening can hardly capture the broad range of compounds present in stormwater and receiving streams. Thus, an additional monitoring proxy that describes the overall chemical load in stormwater is needed. Each chemical in a mixture contributes, albeit with different potency, to cytotoxicity measured by reduction of cell viability after 24h in four human cell lines. Thus, the aim of this study was to investigate the applicability of cytotoxicity as a proxy for the organic contaminant load of rivers during storm events. Field investigations took place in the Ammer River (annual average discharge 0.87 m³ s-1) close to Tübingen, Germany, during intense precipitation events in June 2021. The sampling site was located at the outlet of the gauged catchment (134 km²), thus, integrating inflowing water from all upstream tributaries and sewer overflows. During storm events, high-resolution temporal monitoring of discharge, suspended particles, particle characteristics, as well as dissolved and particle-associated organic contaminants was conducted using both chemical analyses and cell-based in vitro bioassays. The cytotoxicity in the water phase (expressed as toxic units, TU), was similar among the cell lines. The TU flux followed the course of the hydrograph with highest values at the maximum or slightly after the discharge peak. This finding suggests that the chemical load is controlled by the transported volume of water despite the fact that different contaminant sources are likely to contribute to the water flux and pollutant load in the river at different time points of the hydrograph. For the particle-associated cytotoxicity, the TU flux also followed the course of the events suggesting that the particle-associated cytotoxicity in the river is, similar to the water cytotoxicity, controlled by the particle load in the river. This highlights that the cytotoxicity is a suitable proxy to detect mixtures of organic compounds and, thus, assess the chemical load in rivers during storm events.

Rain events may impact the chemical pollution burden in rivers. Forty-four small streams in Germany were profiled during several rain events for the presence of 395 chemicals and five types of mixture effects in in vitro bioassays (cytotoxicity; activation of the estrogen, aryl hydrocarbon, and peroxisome proliferator-activated receptors; and oxidative stress response). While these streams were selected to cover a wide range of agricultural impacts, in addition to the expected pesticides, wastewater-derived chemicals and chemicals typical for street runoff were detected. The unexpectedly high estrogenic effects in many samples indicated the impact by wastewater or overflow of combined sewer systems. The 128 water samples exhibited a high diversity of chemical and effect patterns, even for different rain events at the same site. The detected 290 chemicals explained only a small fraction (

Exposure assessment in in vitro cell-based bioassays is challenging for ionizable organic chemicals (IOCs), because they are present as more than one chemical species in the bioassay medium. Furthermore, compared to neutral organic chemicals, their binding to medium proteins and lipids is driven by more complex molecular interactions. Total medium concentrations (Ctotal,medium) and/or freely dissolved medium concentrations (Cfree,medium) were determined for one neutral chemical and 14 IOCs (acids, bases, multifunctional) at concentrations relevant for determination of cytotoxicity and effect. Cfree,medium was measured in two in vitro bioassays at the time of dosing and after 24 h of incubation using solid-phase microextraction. Cfree,medium was maximally 1.7 times lower than the nominal concentrations (Cnom) for the hydrophilic chemicals (caffeine and lamotrigine). For the organic acids (naproxen, ibuprofen, warfarin, and diclofenac), Cfree,medium was by a factor of 4 lower than Cnom at high concentrations, but the ratio was much higher at low concentrations, indicating a nonlinear binding behavior. The experimental Cfree,medium was also compared with Cfree,medium predicted with a mass balance model accounting for binding to medium proteins and lipids. The mass balance model performed well for five of the test chemicals (within a factor of 10), but it underestimated Cfree,medium by up to a factor of 1200 for chemicals that showed nonlinear binding to medium components. These findings emphasize that experimental exposure assessment is required for improved understanding of in vitro toxicity data.

High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell line...

Extraction of chemicals from biota leads to co-extraction of lipids. When dosing such extracts into in vitro bioassays, co-dosed lipids act as an additional phase that can reduce the bioavailability of the chemicals and the apparent sensitivity of the assay. Equilibrium partitioning between medium, cells, and co-dosed lipids was described with an existing equilibrium partitioning model for cell-based bioassays extended by an additional lipid phase. We experimentally investigated the influence of co-dosed lipids on the effects elicited by four test chemicals of different hydrophobicity in two bioassays, indicative of the aryl hydrocarbon receptor and oxidative stress response (AREc32). The partitioning model explained the effect of the test chemicals in the presence of spiked triolein within a factor of 0.33−5.83 between the measured and predicted effect concentration (EC). We applied the model to marine mammal blubber extracted with silicone. Extracts dosed in the AREc32 bioassay showed a linear increase of apparent EC with increasing lipid fraction. The partitioning model was used to interpret the role of the co-extracted lipid. A quantitative lipid correction of bioassay results in the presence of co-dosed lipids was possible for known compounds and defined mixtures, while we could only estimate a range for mixtures of unknown chemicals.

Surface waters feeding water treatment plants (WTPs) can contain organic micropollutants, which are typically removed during treatment, while disinfection by-products (DBPs) can form after disinfection. The complex mixtures of chemicals in drinking water imply that targeted chemical analysis cannot capture all chemicals present, though in vitro bioassays can be applied alongside chemical analysis to monitor the total chemical burden. The current study applied bioassays indicative of hormone receptor-mediated effects to evaluate micropollutant removal during treatment, while bioassays indicative of adaptive stress responses and mutagenicity were applied to assess DBP formation. Water was extracted with solid-phase extraction from three WTPs using different treatment processes including biological treatment, nanofiltration and ozonation. Of the studied hormone receptors, only estrogenic activity was detected in the source waters feeding the WTPs, with all treatment processes able to remove estrogenic activity in the produced water completely or just above the detection limit. The oxidative stress response and NF-κB response for inflammation were detected in both source and treated water samples, with formed DBPs contributing to the increase in oxidative stress response. None of the samples induced the p53 response for genotoxicity or had a response in the Ames mutagenicity assay. The effects in the produced water were compared to effect-based trigger values (EBT) for activation of estrogenic activity and oxidative stress response, with the observed effect over 10 times lower than the available EBTs. This emphasises the high quality of the produced drinking water and the value of applying in vitro bioassays for water quality monitoring.

Naturally occurring compounds represent a vast pool of pharmacologically active entities. One of such compounds is andrographolide, which is endowed with many beneficial properties, including the activity against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). To initiate a drug repurposing or hit optimization campaign, it is imperative to unravel the primary mechanism(s) of the antiviral action of andrographolide. Here, we showed by means of a reporter gene assay that andrographolide exerts its anti-SARS-CoV-2 effects by inhibiting the interaction between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2-related factor 2 (NRF2) causing NRF2 upregulation. Moreover, we demonstrated that subtle structural modifications of andrographolide could lead to derivatives with stronger on-target activities and improved physicochemical properties. Our results indicate that further optimization of this structural class is warranted to develop novel COVID-19 therapies.

In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. ‘host shutoff factor’) as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.

Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analysed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and a patient-near replication system using human precision-cut lung slices by two orders of magnitude. While the compounds did not interfere with viral entry, it led to lower viral RNA expression after 24 h, indicating that post-entry pathways were inhibited by the compounds.

Background Prevalence of cognitive decline and dementia is rising globally, with more than 10 million new cases every year. These conditions cause a significant burden for individuals, their caregivers, and health care systems. As no causal treatment for dementia exists, prevention of cognitive decline is of utmost importance. Notably, alcohol is among the most significant modifiable risk factors for cognitive decline. Methods Longitudinal data across 15 years on 6,967 individuals of the Survey of Health, Ageing and Retirement in Europe were used to analyze the effect of alcohol consumption and further modifiable (i.e., smoking, depression, and educational obtainment) and non-modifiable risk factors (sex and age) on cognitive functioning (i.e., memory and verbal fluency). For this, a generalized estimating equation linear model was estimated for every cognitive test domain assessed. Results Consistent results were revealed in all three regression models: A nonlinear association between alcohol consumption and cognitive decline was found—moderate alcohol intake was associated with overall better global cognitive function than low or elevated alcohol consumption or complete abstinence. Furthermore, female sex and higher educational obtainment were associated with better cognitive function, whereas higher age and depression were associated with a decline in cognitive functioning. No significant association was found for smoking. Conclusion Our data indicate that alcohol use is a relevant risk factor for cognitive decline in older adults. Furthermore, evidence-based therapeutic concepts to reduce alcohol consumption exist and should be of primary interest in prevention measures considering the aging European population.

Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner.

Substituted para-benzoquinones and hydroquinones are ubiquitous transformation products that arise during oxidative water treatment of phenolic precursors, for example through ozonation or chlorination. The benzoquinone structural motive is associated with mutagenicity and carcinogenicity, and also with induction of the oxidative stress response through the Nrf2 pathway. For either endpoint, toxicological data for differently substituted compounds are scarce. In this study, oxidative stress response, as indicated by the AREc32 in vitro bioassay, was induced by differently substituted para-benzoquinones, but also by the corresponding hydroquinones. Bioassays that indicate defense against genotoxicity (p53RE-bla) and DNA repair activity (UmuC) were not activated by these compounds. Stability tests conducted under incubation conditions, but in the absence of cell lines, showed that tested para-benzoquinones reacted rapidly with constituents of the incubation medium. Compounds were abated already in phosphate buffer, but even faster in biological media, with reactions attributed to amino- and thiol-groups of peptides, proteins, and free amino acids. The products of these reactions were often the corresponding substituted hydroquinones. Conversely, differently substituted hydroquinones were quantitatively oxidized to p-benzoquinones over the course of the incubation. The observed induction of the oxidative stress response was attributed to hydroquinones that are presumably oxidized to benzoquinones inside the cells. Despite the instability of the tested compounds in the incubation medium, the AREc32 in vitro bioassay could be used as an unspecific sum parameter to detect para-benzoquinones and hydroquinones in oxidatively treated waters. ISSN:0043-1354 ISSN:1879-2448

Most studies using high-throughput cell-based bioassays tested chemicals up to a certain fixed concentration. It would be more appropriate to test up to concentrations predicted to elicit baseline toxicity because this is the minimal toxicity of every chemical. Baseline toxicity is also called narcosis and refers to nonspecific intercalation of chemicals in biological membranes, leading to loss of membrane structure and impaired functioning of membrane-related processes such as mitochondrial respiration. In cells, baseline toxicity manifests as cytotoxicity, which was quantified by a robust live-cell imaging method. Inhibitory concentrations for baseline toxicity varied by orders of magnitude between chemicals and were described by a simple quantitative structure activity relationship (QSAR) with the liposome-water partition constant as a sole descriptor. The QSAR equations were remarkably similar for eight reporter gene cell lines of different cellular origin, six of which were used in Tox21. Mass-balance models indicated constant critical membrane concentrations for all cells and all chemicals with a mean of 69 mmol·kg(95% CI: 49-89), which is in the same range as for bacteria and aquatic organisms and consistent with the theory of critical membrane burden of narcosis. The challenge of developing baseline QSARs for cell lines is that many confirmed baseline toxicants are rather volatile. We deduced from cytotoxicity experiments with semi-volatile chemicals that only chemicals with medium-air partition constants >10,000 L/L can be tested in standard robotic setups without appreciable loss of effect. Chemicals just below that cutoff showed crossover effects in neighboring wells, whereas the effects of chemicals with lower medium-air partition constants were plainly lost. Applying the "volatility cut-off" to >8000 chemicals tested in Tox21 indicated that approximately 20% of Tox21 chemicals could have partially been lost during the experiments. We recommend applying the baseline QSARs together with volatility cut-offs for experimental planning of reporter gene assays, that is, to dose only chemicals with medium-air partition constants >10,000 at concentrations up to the baseline toxicity level.

Improved understanding of chemical exposure in in vitro bioassays is required for quantitative in vitro–in vivo extrapolation (QIVIVE). In this study, we quantified freely dissolved concentrations in medium sampled from in vitro cell-based bioassays (Cfree,medium) for nine chemicals with different hydrophobicity and speciation at the time point of dosing and after an incubation period of 24 h using solid-phase microextraction. The chemicals were tested in two reporter gene assays, the AREc32 assay indicative of the oxidative stress response and the PPARγ-GeneBLAzer assay that responds to chemicals which bind to the peroxisome proliferator-activated receptor gamma. For seven of the nine chemicals, Cfree,medium did not change significantly over time in both assays and the experimentally determined Cfree,medium generally agreed well with predictions of a mass balance model that describes the partitioning between proteinaceous and lipidous medium constituents, cells and the aqueous phase. Two chemicals showed a decrease of Cfree,medium in the AREc32 assay over time that was probably caused by cellular metabolism. Furthermore, Cfree,medium of the acidic chemical diclofenac deviated from the model predictions by more than a factor of 10 at higher concentrations, which indicates nonlinear binding and saturation of the medium proteins. Bioassay results are typically reported as nominal effect concentrations (ECnom), although it is established that freely dissolved effect concentrations (ECfree) are a better measure for the bioavailable dose and the method developed here provides a simple experimental approach to measure and model ECfree in in vitro bioassay for improved QIVIVE models.

Some environmental pollutants impair mitochondria, which are of vital importance as energy factories in eukaryotic cells. Mitochondrial toxicity was quantified by measuring the change of the oxygen consumption rate (OCR) of HepG2 cells with the Agilent Seahorse XFe 96 Analyzer. Various mechanisms of mitochondrial toxicity, including inhibition of the electron transport chain or adenosine triphosphate (ATP) synthase as well as uncoupling of oxidative phosphorylation, were differentiated by dosing the sample in parallel with reference compounds following the OCR over time. These time-OCR traces were used to derive effect concentrations for 10% inhibition of the electron transport chain or 10% of uncoupling. The low effect level of 10% was necessary because environmental mixtures contain thousands of chemicals; only few of them interfere with mitochondria, but the others cause cytotoxicity. The OCR bioassay was validated with environmental pollutants of known mechanism of mitochondrial toxicity. Binary mixtures of uncouplers or inhibitors acted according to the mixture model of concentration addition. Uncoupling and/or inhibitory effects were detected in extracts of river water samples without apparent cytotoxicity. Uncoupling effects could only be quantified in water samples if inhibitory effects occurred at lower concentrations because no uncoupling can be detected without an appreciable membrane potential built up. The OCR bioassay can thus complement chemical analysis and in vitro bioassays for monitoring micropollutants in water. Environ Toxicol Chem 2019;00:1-12. © 2019 SETAC.

International audience; Seven treatment wetlands and a municipal wastewater treatment plant (WWTP) were weekly monitored over the course of one year for removal of conventional wastewater parameters, selected micropollutants (caffeine, ibuprofen, naproxen, benzotriazole, diclofenac, acesulfame, and carbamazepine) and biological effects. The treatment wetland designs investigated include a horizontal subsurface flow (HF) wetland and a variety of wetlands with intensification (aeration, two-stages, or reciprocating flow). Complementary to the common approach of analyzing individual chemicals, in vitro bioassays can detect the toxicity of a mixture of known and unknown components given in a water sample. A panel of five in vitro cell-based reporter gene bioassays was selected to cover environmentally relevant endpoints (AhR: indicative of activation of the aryl hydrocarbon receptor; PPARγ: binding to the peroxisome proliferator-activated receptor gamma; ERα: activation of the estrogen receptor alpha; GR: activation of the glucocorticoid receptor; oxidative stress response). While carbamazepine was persistent in the intensified treatment wetlands, mean monthly mass removal of up to 51% was achieved in the HF wetland. The two-stage wetland system showed highest removal efficacy for all biological effects (91% to >99%). The removal efficacy for biological effects ranged from 56% to 77% for the HF wetland and 60% to 99% for the WWTP. Bioanalytical equivalent concentrations (BEQs) for AhR, PPARγ, and oxidative stress response were often below the recommended effect-based trigger (EBT) values for surface water, indicating the great benefit for using nature-based solutions for water treatment. Intensified treatment wetlands remove both individual micropollutants and mixture effects more efficiently than conventional (non-aerated) HF wetlands, and in some cases, the WWTP.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17β-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17β-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75–304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays – based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions., Graphical abstract Unlabelled Image, Highlights • Estrogens are active at concentrations undetectable by chemical analysis. • Bioassays detected estrogenic effects by other EDC than only hormones in water. • Estrogenicity bioassays have different responsiveness to specific compounds. • Some bioassays detected the additive effects of estrogens. • Reference materials enlightened the ability of bioassays to detect combined effects.

The identification of mixture risk drivers is a great challenge for sediment assessment, especially when taking bioavailability into consideration. The bioavailable portion, which comprises the organic contaminants in pore water and the ones bound to organic carbon, was accessed by equilibrium partitioning to polydimethylsiloxane (PDMS). The exhaustive solvent and PDMS extracts were toxicologically characterized with a battery of in vitro reporter gene assays and chemically analyzed with liquid and gas chromatography coupled to high-resolution mass spectrometry. The bioavailable fractions of mixture effects and individual chemicals were mostly lower than 0.1, indicating that more than 90% of the substances are strongly bound and would not pose an immediate risk but could potentially be remobilized in the long term. Despite 655 organic chemicals analyzed, only 0.1-28% of the observed biological effects was explained by the detected compounds in whole sediments, while 0.009-3.3% was explained by bioavailable chemicals. The mixture effects were not only dominated by legacy pollutants (e.g., polycyclic aromatic hydrocarbons (PAHs) in the bioassay for activation of the aryl-hydrocarbon receptor (AhR) and oxidative stress response (AREc32)) but also by present-use chemicals (e.g., plastic additives for binding to the peroxisome proliferator-activated receptor γ (PPARγ)), with different fingerprints between whole sediments and bioavailable extracts. Our results highlight the necessity to involve different bioassays with diverse effect profiles and broader selection of contaminants along with bioavailability for the risk assessment of chemical mixtures in sediments.

Background: High-throughput screening of chemicals with in vitro reporter gene assays in Tox21 has produced a large database on cytotoxicity and specific modes of action. However, the validity of some of the reported activities is questionable due to the “cytotoxicity burst,” which refers to the supposition that many stress responses are activated in a nonspecific way at concentrations close to cell death. Objectives: We propose a pragmatic method to identify whether reporter gene activation is specific or cytotoxicity-triggered by comparing the measured effects with baseline toxicity. Methods: Baseline toxicity, also termed narcosis, is the minimal toxicity any chemical causes. Quantitative structure–activity relationships (QSARs) developed for baseline toxicity in mammalian reporter gene cell lines served as anchors to define the chemical-specific threshold for the cytotoxicity burst and to evaluate the degree of specificity of the reporter gene activation. Measured 10% effect concentrations were related to measured or QSAR-predicted 10% cytotoxicity concentrations yielding specificity ratios (SR). We applied this approach to our own experimental data and to ∼8,000 chemicals that were tested in six of the high-throughput Tox21 reporter gene assays. Results: Confirmed baseline toxicants activated reporter gene activity around cytotoxic concentrations triggered by the cytotoxicity burst. In six Tox21 assays, 37%–87% of the active hits were presumably caused by the cytotoxicity burst (SR

BACKGROUND Nutrition apps are a prototypical mobile health (mHealth) technology supporting healthy eating behavior that are seen as promising tools for health promotion by policy makers. Although nutrition apps are increasingly popular, wide-spread adoption is yet to be achieved. Hence, profound knowledge regarding factors motivating and hindering (long-term) nutrition app use is crucial for developing design guidelines aiming at supporting uptake and prolonged use of nutrition apps. OBJECTIVE In this scoping review, we synthesized the literature on barriers to and facilitators for nutrition app use across disciplines including empirical qualitative and quantitative studies with current users, ex-users, and/or non-users of nutrition apps. METHODS A systematic literature search including six databases as well as backward and forward citation search was conducted. Search strategy, inclusion and exclusion criteria, and the planned data extraction process were preregistered. All empirical qualitative and quantitative studies published in German or English were eligible for inclusion if they examined adults or adolescents (aged 13 to 18) who were either current users, ex-users and/ or non-users of nutrition apps. Based on qualitative content analysis, extracted individual barriers and facilitators were grouped into categories. RESULTS Twenty-eight publications were identified as eligible. A framework with a three-level hierarchy was designed which grouped 326 individual barriers and facilitators into 21 sub-categories, twelve categories, and four clusters that focus on either the individual user (goals, goal attainment, goal abandonment, personal living conditions, lack of knowledge or skill, lack or loss of motivation, habit), different aspects of the app and the smartphone (features, usability of the app or food database, technical issues, data security, accuracy/trustworthiness, costs), positive and negative outcomes of nutrition app use, or interactions between the user and their social environment. CONCLUSIONS The resulting conceptual framework underlines a pronounced diversity of reasons for (not) using nutrition apps indicating that there is no “one-size-fits-all” approach for uptake and prolonged use of nutrition aps. Hence, tailoring nutrition apps to needs of specific user groups seems promising for increasing engagement.

Exposure assessment in