Reporting and reproducibility: Proteomics of fish models in environmental toxicology and ecotoxicology.

Environmental toxicology and ecotoxicology research efforts are employing proteomics with fish models as New Approach Methodologies, along with in silico, in vitro and other omics techniques to elucidate hazards of toxicants and toxins. We performed a critical review of toxicology studies with fish models using proteomics and reported fundamental parameters across experimental design, sample preparation, mass spectrometry, and bioinformatics of fish, which represent alternative vertebrate models in environmental toxicology, and routinely studied animals in ecotoxicology. We observed inconsistencies in reporting and methodologies among experimental designs, sample preparations, data acquisitions and bioinformatics, which can affect reproducibility of experimental results. We identified a distinct need to develop reporting guidelines for proteomics use in environmental toxicology and ecotoxicology, increased QA/QC throughout studies, and method optimization with an emphasis on reducing inconsistencies among studies. Several recommendations are offered as logical steps to advance development and application of this emerging research area to understand chemical hazards to public health and the environment.

Comparison of Two Estrogen Chemically Activated Luciferase Expression Cell Bioassays to Liquid Chromatography-Mass Spectrometry for Quantifying Estrone in Water Samples.

Chemically activated luciferase expression (CALUX) cell bioassays are popular tools for assessing endocrine activity of chemicals such as certain environmental contaminants. Although activity equivalents can be obtained from CALUX analysis, directly comparing these equivalents to those obtained from analytical chemistry methods can be problematic because of the complexity of endocrine active pathways. We explored the suitability of two estrogen CALUX bioassays (the Organisation for Economic Co-operation and Development-approved VM7Luc4E2 cell bioassay and the VM7LucERßc9 cell bioassay) for quantitation of estrogen. Quadrupole-time of flight ultraperformance liquid chromatography-mass spectrometry (LC/MS) was selected as a comparative method. Regression analysis of measured estrone (E1) calibration samples showed all three methods to be highly predictive of nominal concentrations (p ≤ 7.5 × 10-51 ). Extracts of water sampled from laboratory dilutor tanks containing E1 at 0, 20, and 200 ng/L alone and in combination with atrazine were selected to test the quantitative capabilities of the CALUX assays. Process controls (0 and 100 ng E1/L) and a separate E1 standard (10 ng/ml, used to prepare the E1 process control) were also tested. Levels of E1 determined by LC/MS analysis and bioanalytical equivalents (ng E1/L) determined by CALUX analyses were comparable except in certain instances where the samples required dilution prior to CALUX analyses (e.g., the E1 process control and E1 standard). In those instances, measurements by CALUX were slightly but significantly decreased relative to LC/MS. Atrazine had no effect on the ability of either LC/MS or the CALUX bioassays to quantify E1. The present study illustrates the CALUX bioassays as successful in quantifying an estrogen in simple water samples and further characterizes their utility for screening. Environ Toxicol Chem 2023;42:333-339. Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

Experimental arena size alters larval zebrafish photolocomotor behaviors and influences bioactivity responses to a model neurostimulant.

Zebrafish behavior is increasingly common in biomedical and environmental studies of chemical bioactivity. Multiple experimental arena sizes have been used to measure photolocomotion in zebrafish depending on age, endpoints observed, and instrumentation, among other factors. However, the extent to which methodological parameters may influence naïve behavioral performance and detection of behavioral changes is poorly understood. Here we measured photolocomotion and behavioral profiles of naïve larval zebrafish across arena sizes. We then performed concentration response studies with the model neurostimulant caffeine, again across various arena dimensions. We found total swimming distance of unexposed fish to increase logarithmically with arena size, which as related to circumference, area, and volume. Photomotor response during light/dark transitions also increased with arena size. Following caffeine exposure, total distance travelled was significantly (p < 0.001) affected by well size, caffeine treatment (p < 0.001), and the interaction of these two experimental factors (p < 0.001). In addition, behavioral response profiles showed differences between 96 well plates and larger well sizes. Biphasic response, with stimulation at lower concentrations and refraction at the highest concentration, was observed in dark conditions for the 96 well size only, though almost no effects were identified in the light. However, swimming behavior was significantly (p < 0.1) altered in the highest studied caffeine treatment level in larger well sizes during both light and dark periods. Our results indicate zebrafish swim more in larger arenas and arena size influences behavioral response profiles to caffeine, though differences were mostly observed between very small and large arenas. Further, careful consideration should be given when choosing arena size, because small wells may lead to restriction, while larger wells may differentially reflect biologically relevant effects. These findings can improve comparability among experimental designs and demonstrates the importance of understanding confounding methodological variables.

Quantitative Reverse Transcription PCR Surveillance of SARS-CoV-2 Variants of Concern in Wastewater of Two Counties in Texas, United States.

After its emergence in late November/December 2019, the severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) rapidly spread globally. Recognizing that this virus is shed in feces of individuals and that viral RNA is detectable in wastewater, testing for SARS-CoV-2 in sewage collections systems has allowed for the monitoring of a community's viral burden. Over a 9 month period, the influents of two regional wastewater treatment facilities were concurrently examined for wild-type SARS-CoV-2 along with variants B.1.1.7 and B.1.617.2 incorporated as they emerged. Epidemiological data including new confirmed COVID-19 cases and associated hospitalizations and fatalities were tabulated within each location. RNA from SARS-CoV-2 was detectable in 100% of the wastewater samples, while variant detection was more variable. Quantitative reverse transcription PCR (RT-qPCR) results align with clinical trends for COVID-19 cases, and increases in COVID-19 cases were positively related with increases in SARS-CoV-2 RNA load in wastewater, although the strength of this relationship was location specific. Our observations demonstrate that clinical and wastewater surveillance of SARS-CoV-2 wild type and constantly emerging variants of concern can be combined using RT-qPCR to characterize population infection dynamics. This may provide an early warning for at-risk communities and increases in COVID-19 related hospitalizations.