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In this study, we comprehensively evaluate chloride- and ionic-strength-mediated changes in the physical morphology, dissolution, and bacterial toxicity of silver nanoparticles (AgNPs), which are one of the most-used nanomaterials. The findings isolate the impact of ionic strength from that of chloride concentration. As ionic strength increases, AgNP aggregation likewise increases (such that the hydrodynamic radius [HR] increases), fractal dimension (Df) strongly decreases (providing increased available surface relative to suspensions with higher Df), and the release of Ag(aq) increases. With increased Ag+ in solution, Escherichia coli demonstrates reduced tolerance to AgNP exposure (i.e., toxicity increases) under higher ionic strength conditions. As chloride concentration increases, aggregates are formed (HR increases) but are dominated by AgCl0 (s) bridging of AgNPs; relatedly, Df increases. Furthermore, AgNP dissolution strongly increases under increased chloride conditions, but the dominant, theoretical, equilibrium aqueous silver species shift to negatively charged AgClx(x-1)- species, which appear to be less toxic to E. coli. Thus, E. coli demonstrates increased tolerance to AgNP exposure under higher chloride conditions (i.e., toxicity decreases). Expression measurements of katE, a gene involved in catalase production to alleviate oxidative stress, support oxidative stress in E. coli as a result of Ag+ exposure. Overall, our work indicates that the environmental impacts of AgNPs must be evaluated under relevant water chemistry conditions. [ABSTRACT FROM AUTHOR]

High-throughput transcriptomics (HTTr) uses gene expression profiling to characterize the biological activity of chemicals in in vitro cell-based test systems. As an extension of a previous study testing 44 chemicals, HTTr was used to screen an additional 1,751 unique chemicals from the EPA's ToxCast collection in MCF7 cells using 8 concentrations and an exposure duration of 6 h. We hypothesized that concentration-response modeling of signature scores could be used to identify putative molecular targets and cluster chemicals with similar bioactivity. Clustering and enrichment analyses were conducted based on signature catalog annotations and ToxPrint chemotypes to facilitate molecular target prediction and grouping of chemicals with similar bioactivity profiles. Enrichment analysis based on signature catalog annotation identified known mechanisms of action (MeOAs) associated with well-studied chemicals and generated putative MeOAs for other active chemicals. Chemicals with predicted MeOAs included those targeting estrogen receptor (ER), glucocorticoid receptor (GR), retinoic acid receptor (RAR), the NRF2/KEAP/ARE pathway, AP-1 activation, and others. Using reference chemicals for ER modulation, the study demonstrated that HTTr in MCF7 cells was able to stratify chemicals in terms of agonist potency, distinguish ER agonists from antagonists, and cluster chemicals with similar activities as predicted by the ToxCast ER Pathway model. Uniform manifold approximation and projection (UMAP) embedding of signature-level results identified novel ER modulators with no ToxCast ER Pathway model predictions. Finally, UMAP combined with ToxPrint chemotype enrichment was used to explore the biological activity of structurally related chemicals. The study demonstrates that HTTr can be used to inform chemical risk assessment by determining in vitro points of departure, predicting chemicals' MeOA and grouping chemicals with similar bioactivity profiles. [ABSTRACT FROM AUTHOR]

Hydraulic performance issues in drinking‐water biofilters have sometimes been associated with phosphorus limitation and increased production of extracellular polymeric substances in previous bench‐scale studies. However, field studies utilizing phosphorus supplementation to improve biofilter hydraulic performance have produced mixed results. Here, we determined the ratio of activities for phosphatase to glycosidase (PHO:GLY), which are enzymes involved in acquiring orthophosphate and biodegradable organic carbon from complex organic substrates, to assess phosphorus limitation in 21 pilot‐ and full‐scale biofilters. Supplementation of the pilot‐scale biofilter influents with 37 μg/L orthophosphate‐P reduced the PHO:GLY from 1.8–40.3 (mean 14.8) to 0.3–15.9 (mean 5.3), demonstrating that increased orthophosphate availability decreases PHO:GLY. In the absence of phosphorus supplementation, the PHO:GLY of the pilot‐ and full‐scale biofilters ranged from 0.3 to 40.3 (mean 10.1), and no hydraulic performance issues were noted. Thus, severe phosphorus limitation appears uncommon in the field, suggesting that phosphorus supplementation is unlikely to improve hydraulic performance in typical drinking water biofilters. [ABSTRACT FROM AUTHOR]

Using in vitro data to estimate point of departure (POD) values is an essential component of new approach methodologies (NAMs)-based chemical risk assessments. In this case study, we evaluated a NAM for hepatotoxicity based on rat primary hepatocytes, high-content imaging (HCI), and toxicokinetic modeling. First, we treated rat primary hepatocytes with 10 concentrations (0.2–100 µM) of 51 chemicals that produced hepatotoxicity in repeat-dose subchronic and chronic exposures. Second, we used HCI to measure endoplasmic reticulum stress, mitochondrial function, lysosomal mass, steatosis, apoptosis, DNA texture, nuclear size, and cell number at 24, 48, and 72 h and calculated concentrations at 50% maximal activity (AC50). Third, we estimated administered equivalent doses (AEDs) from AC50 values using toxicokinetic modeling. AEDs using physiologically based toxicokinetic models were 4.1-fold (SD 6.3) and 8.1-fold (SD 15.5) lower than subchronic and chronic lowest observed adverse effect levels (LOAELs), respectively. In contrast, AEDs from ToxCast and Tox21 assays were 89.8-fold (SD 149.5) and 168-fold (SD 323.7) lower than subchronic and chronic LOAELs. Individual HCI endpoints also estimated AEDs for specific hepatic lesions that were lower than in vivo PODs. Lastly, AEDs were similar for different in vitro exposure durations, but steady-state toxicokinetic models produced 7.6-fold lower estimates than dynamic physiologically based ones. Our findings suggest that NAMs from diverse cell types provide conservative estimates of PODs. In contrast, NAMs based on the same species and cell type as the adverse outcome may produce estimates closer to the traditional in vivo PODs. [ABSTRACT FROM AUTHOR]

New approach methodologies (NAMs) aim to accelerate the pace of chemical risk assessment while simultaneously reducing cost and dependency on animal studies. High Throughput Transcriptomics (HTTr) is an emerging NAM in the field of chemical hazard evaluation for establishing in vitro points-of-departure and providing mechanistic insight. In the current study, 1201 test chemicals were screened for bioactivity at eight concentrations using a 24-h exposure duration in the human- derived U-2 OS osteosarcoma cell line with HTTr. Assay reproducibility was assessed using three reference chemicals that were screened on every assay plate. The resulting transcriptomics data were analyzed by aggregating signal from genes into signature scores using gene set enrichment analysis, followed by concentration-response modeling of signatures scores. Signature scores were used to predict putative mechanisms of action, and to identify biological pathway altering concentrations (BPACs). BPACs were consistent across replicates for each reference chemical, with replicate BPAC standard deviations as low as 5.6 × 10−3 μM, demonstrating the internal reproducibility of HTTr-derived potency estimates. BPACs of test chemicals showed modest agreement (R2 = 0.55) with existing phenotype altering concentrations from high throughput phenotypic profiling using Cell Painting of the same chemicals in the same cell line. Altogether, this HTTr based chemical screen contributes to an accumulating pool of publicly available transcriptomic data relevant for chemical hazard evaluation and reinforces the utility of cell based molecular profiling methods in estimating chemical potency and predicting mechanism of action across a diverse set of chemicals. • 1201 chemicals screened with high throughput transcriptomics in U-2 OS cells. • Potency estimates for reference chemical replicates were generally reproducible. • 97.5% of chemicals have at least one concentration responsive signature. • Predicted NR3C1, RARA, and PGR agonists using signature similarity. • Potency estimates show modest agreement with cell painting derived potencies. [ABSTRACT FROM AUTHOR]

'Cell Painting' is an imaging-based high-throughput phenotypic profiling (HTPP) method in which cultured cells are fluorescently labeled to visualize subcellular structures (i.e., nucleus, nucleoli, endoplasmic reticulum, cytoskeleton, Golgi apparatus / plasma membrane and mitochondria) and to quantify morphological changes in response to chemicals or other perturbagens. HTPP is a high-throughput and cost-effective bioactivity screening method that detects effects associated with many different molecular mechanisms in an untargeted manner, enabling rapid in vitro hazard assessment for thousands of chemicals. Here, 1201 chemicals from the ToxCast library were screened in concentration-response up to ∼100 μM in human U-2 OS cells using HTPP. A phenotype altering concentration (PAC) was estimated for chemicals active in the tested range. PACs tended to be higher than lower bound potency values estimated from a broad collection of targeted high-throughput assays, but lower than the threshold for cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was used to estimate administered equivalent doses (AEDs) based on PACs for comparison to human exposure predictions. AEDs for 18/412 chemicals overlapped with predicted human exposures. Phenotypic profile information was also leveraged to identify putative mechanisms of action and group chemicals. Of 58 known nuclear receptor modulators, only glucocorticoids and retinoids produced characteristic profiles; and both receptor types are expressed in U-2 OS cells. Thirteen chemicals with profile similarity to glucocorticoids were tested in a secondary screen and one chemical, pyrene, was confirmed by an orthogonal gene expression assay as a novel putative GR modulating chemical. Most active chemicals demonstrated profiles not associated with a known mechanism-of-action. However, many structurally related chemicals produced similar profiles, with exceptions such as diniconazole, whose profile differed from other active conazoles. Overall, the present study demonstrates how HTPP can be applied in screening-level chemical assessments through a series of examples and brief case studies. • The percentage of ToxCast chemicals active in Cell Painting was 49.3%. • Pharmaceuticals and pesticides were more active than food additives and inerts. • 4.4% of chemicals were bioactive at predicted human exposure concentrations. • Pyrene produced a profile similar to known glucocorticoid receptor agonists. • Diniconazole bioactivity differed from other conazoles and structurally related chemicals. [ABSTRACT FROM AUTHOR]