Proteome changes in larval zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) exposed to (±) anatoxin-a.

Anatoxin-a and its analogues are potent neurotoxins produced by several genera of cyanobacteria. Due in part to its high toxicity and potential presence in drinking water, these toxins pose threats to public health, companion animals and the environment. It primarily exerts toxicity as a cholinergic agonist, with high affinity at neuromuscular junctions, but molecular mechanisms by which it elicits toxicological responses are not fully understood. To advance understanding of this cyanobacteria, proteomic characterization (DIA shotgun proteomics) of two common fish models (zebrafish and fathead minnow) was performed following  (±) anatoxin-a exposure. Specifically, proteome changes were identified and quantified in larval fish exposed for 96 h (0.01-3 mg/L (±) anatoxin-a and caffeine (a methodological positive control) with environmentally relevant treatment levels examined based on environmental exposure distributions of surface water data. Proteomic concentration - response relationships revealed 48 and 29 proteins with concentration - response relationships curves for zebrafish and fathead minnow, respectively. In contrast, the highest number of differentially expressed proteins (DEPs) varied between zebrafish (n = 145) and fathead minnow (n = 300), with only fatheads displaying DEPs at all treatment levels. For both species, genes associated with reproduction were significantly downregulated, with pathways analysis that broadly clustered genes into groups associated with DNA repair mechanisms. Importantly, significant differences in proteome response between the species was also observed, consistent with prior observations of differences in response using both behavioral assays and gene expression, adding further support to model specific differences in organismal sensitivity and/or response. When DEPs were read across from humans to zebrafish, disease ontology enrichment identified diseases associated with cognition and muscle weakness consistent with the prior literature. Our observations highlight limited knowledge of how (±) anatoxin-a, a commonly used synthetic racemate surrogate, elicits responses at a molecular level and advances its toxicological understanding.

Influences of 23 different equations used to calculate gene copies of SARS-CoV-2 during wastewater-based epidemiology.

Following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019, the use of wastewater-based surveillance (WBS) has increased dramatically along with associated infrastructure globally. However, due to the global nature of its application, and various workflow adaptations (e.g., sample collection, water concentration, RNA extraction kits), numerous methods for back-calculation of gene copies per volume (gc/L) of sewage have also emerged. Many studies have considered the comparability of processing methods (e.g., water concentration, RNA extraction); however, for equations used to calculate gene copies in a wastewater sample and subsequent influences on monitoring viral trends in a community and its association with epidemiological data, less is known. Due to limited information on how many formulas exist for the calculation of SARS-CoV-2 gene copies in wastewater, we initially attempted to quantify how many equations existed in the referred literature. We identified 23 unique equations, which were subsequently applied to an existing wastewater dataset. We observed a range of gene copies based on use of different equations, along with variability of AUC curve values, and results from correlation and regression analyses. Though a number of individual laboratories appear to have independently converged on a similar formula for back-calculation of viral load in wastewater, and share similar relationships with epidemiological data, differential influences of various equations were observed for variation in PCR volumes, RNA extraction volumes, or PCR assay parameters. Such observations highlight challenges when performing comparisons among WBS studies when numerous methodologies and back-calculation methods exist. To facilitate reproducibility among studies, the different gc/L equations were packaged as an R Shiny app, which provides end users the ability to investigate variability within their datasets and support comparisons among studies.

Big Question to Developing Solutions: A Decade of Progress in the Development of Aquatic New Approach Methodologies from 2012 to 2022.

In 2012, 20 key questions related to hazard and exposure assessment and environmental and health risks of pharmaceuticals and personal care products in the natural environment were identified. A decade later, this article examines the current level of knowledge around one of the lowest-ranking questions at that time, number 19: "Can nonanimal testing methods be developed that will provide equivalent or better hazard data compared with current in vivo methods?" The inclusion of alternative methods that replace, reduce, or refine animal testing within the regulatory context of risk and hazard assessment of chemicals generally faces many hurdles, although this varies both by organism (human-centric vs. other), sector, and geographical region or country. Focusing on the past 10 years, only works that might reasonably be considered to contribute to advancements in the field of aquatic environmental risk assessment are highlighted. Particular attention is paid to methods of contemporary interest and importance, representing progress in (1) the development of methods which provide equivalent or better data compared with current in vivo methods such as bioaccumulation, (2) weight of evidence, or (3) -omic-based applications. Evolution and convergence of these risk assessment areas offer the basis for fundamental frameshifts in how data are collated and used for the protection of taxa across the breadth of the aquatic environment. Looking to the future, we are at a tipping point, with a need for a global and inclusive approach to establish consensus. Bringing together these methods (both new and old) for regulatory assessment and decision-making will require a concerted effort and orchestration. Environ Toxicol Chem 2024;43:559-574. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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.

Influence of storage conditions and multiple freeze-thaw cycles on N1 SARS-CoV-2, PMMoV, and BCoV signal.

Wastewater-based epidemiology/wastewater-based surveillance (WBE/WBS) continues to serve as an effective means of monitoring various diseases, including COVID-19 and the emergence of SARS-CoV-2 variants, at the population level. As the use of WBE expands, storage conditions of wastewater samples will play a critical role in ensuring the accuracy and reproducibility of results. In this study, the impacts of water concentration buffer (WCB), storage temperature, and freeze-thaw cycles on the detection of SARS-CoV-2 and other WBE-related gene targets were examined. Freeze-thawing of concentrated samples did not significantly affect (p > 0.05) crossing/cycle threshold (Ct) value for any of the gene targets studied (SARS-CoV-2 N1, PMMoV, and BCoV). However, use of WCB during concentration resulted in a significant (p < 0.05) decrease in Ct for all targets, and storage at -80 °C (in contrast to -20 °C) appeared preferable for wastewater storage signal stability based on decreased Ct values, although this was only significantly different (p < 0.05) for the BCoV target. Interestingly, when Ct values were converted to gene copies per influent sample, no significant differences (p > 0.05) were observed in any of the targets examined. Stability of RNA targets in concentrated wastewater against freeze-thaw degradation supports archiving of concentrated samples for use in retrospective examination of COVID-19 trends and tracing SARS-CoV-2 variants and potentially other viruses, and provides a starting point for establishing a consistent procedure for specimen collection and storage for the WBE/WBS community.

Comparative Analysis of RNA-Extraction Approaches and Associated Influences on RT-qPCR of the SARS-CoV-2 RNA in a University Residence Hall and Quarantine Location.

Wastewater-based epidemiology (WBE) provides an early warning and trend analysis approach for determining the presence of COVID-19 in a community and complements clinical testing in assessing the population level, even as viral loads fluctuate. Here, we evaluate combinations of two wastewater concentration methods (i.e., ultrafiltration and composite supernatant-solid), four pre-RNA extraction modifications, and three nucleic acid extraction kits using two different wastewater sampling locations. These consisted of a quarantine facility containing clinically confirmed COVID-19-positive inhabitants and a university residence hall. Of the combinations examined, composite supernatant-solid with pre-RNA extraction consisting of water concentration and RNA/DNA shield performed the best in terms of speed and sensitivity. Further, of the three nucleic acid extraction kits examined, the most variability was associated with the Qiagen kit. Focusing on the quarantine facility, viral concentrations measured in wastewater were generally significantly related to positive clinical cases, with the relationship dependent on method, modification, kit, target, and normalization, although results were variable-dependent on individual time points (Kendall's Tau-b (τ) = 0.17 to 0.6) or cumulatively (Kendall's Tau-b (τ) = -0.048 to 1). These observations can support laboratories establishing protocols to perform wastewater surveillance and monitoring efforts for COVID-19.

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.

Exploratory analysis of the application of animal reduction approaches in proteomics: How much is enough?

Animal testing has long been the cornerstone of chemical safety assessments, but fish embryo assays represent an alter­native. Omics studies allow the examination of early molecular responses of organisms to environmental stressors, but reduction of animal use within this context has been overlooked. For proteomics, there is significant disparity and vari­ability in the organismal pool size used for studies, ranging from 1-1500 embryos per replicate for zebrafish alone. However, it is unknown if varying sample pool size results in differences in protein identifications. To examine whether the detected proteome changes depend on this variable, 3 pool sizes (5, 10 or 20 embryos or larvae per replicate) were compared using the two most common fish models with an appropriate biological replicate number determined by power analysis (n = 7). Data was acquired using MSe, resulting in 1,946 and 3,172 protein groups identified (1% false discovery rate) for fathead minnow and zebrafish, respectively. Proteins were not differentially expressed among pool sizes, and no significant difference was observed among the identified protein groups. However, for the fathead minnow, a decrease in the number of identified proteins was observed with increasing pool size, while a trend towards an increase in protein identifications was observed in zebrafish between the lowest and highest pool size. Taken together, our observations suggest that a proteome characterization experiment using these fish models can achieve comparable protein identifications using pool sizes of less than 5 organisms per replicate, assuming a protein requirement of 50 µg or less.

Standardizing data reporting in the research community to enhance the utility of open data for SARS-CoV-2 wastewater surveillance.

SARS-CoV-2 RNA detection in wastewater is being rapidly developed and adopted as a public health monitoring tool worldwide. With wastewater surveillance programs being implemented across many different scales and by many different stakeholders, it is critical that data collected and shared are accompanied by an appropriate minimal amount of metainformation to enable meaningful interpretation and use of this new information source and intercomparison across datasets. While some databases are being developed for specific surveillance programs locally, regionally, nationally, and internationally, common globally-adopted data standards have not yet been established within the research community. Establishing such standards will require national and international consensus on what metainformation should accompany SARS-CoV-2 wastewater measurements. To establish a recommendation on minimum information to accompany reporting of SARS-CoV-2 occurrence in wastewater for the research community, the United States National Science Foundation (NSF) Research Coordination Network on Wastewater Surveillance for SARS-CoV-2 hosted a workshop in February 2021 with participants from academia, government agencies, private companies, wastewater utilities, public health laboratories, and research institutes. This report presents the primary two outcomes of the workshop: (i) a recommendation on the set of minimum meta-information that is needed to confidently interpret wastewater SARS-CoV-2 data, and (ii) insights from workshop discussions on how to improve standardization of data reporting.

A comparison of four commercially available RNA extraction kits for wastewater surveillance of SARS-CoV-2 in a college population.

Localized wastewater surveillance has allowed for public health officials to gain a broader understanding of SARS-CoV-2 viral prevalence in the community allowing public health officials time to prepare for impending outbreaks. Given variable levels of virus in the population through public health interventions, proper concentration and extraction of viral RNA is a key step in ensuring accurate detections. With many commercial RNA extraction kits and methodologies available, the performance of 4 different kits were evaluated for SARS-CoV-2 RNA detection in wastewater, specifically focusing on their applicability to lower population densities such as those at university campus dorms. Raw wastewater samples were collected at 4 sites on a college campus over a 24 hour period as a composite sample. Included in these sites was an isolation site that housed students that tested positive for Covid-19 via nasopharyngeal swabs. These samples were analyzed using the following kits: Qiagen All Prep PowerViral DNA/RNA kit, New England BioLabs Monarch RNA MiniPrep Kit, and Zymo Quick RNA-Viral Kit, and the Zymo Quick-RNA Fecal/Soil Microbe MicroPrep Kit. All four sites were processed according to the manufacturer's guidelines. Extractions were then quantified with RT-qPCR one-step reactions using an N2 primer and a linearized plasmid standard. While the Zymo Quick-RNA Fecal/Soil Microbe MicroPrep Kit (also known as the Zymo Environ Water RNA Kit) only recovered approximately 73% (±38%) SARS-CoV-2 RNA compared to the Zymo Quick-RNA Viral kit, it was the most time efficient kit to yield comparable results. This extraction kit had a cumulative processing time of approximately 5 h compared, while the other three kits had processing times between approximately 9 and 9.5 h. Based on the current research, the most effective kits for smaller population densities are pellet based and include a homogenization, inhibitor removal, and RNA preservation step.

Differential influences of (±) anatoxin-a on photolocomotor behavior and gene transcription in larval zebrafish and fathead minnows.

BACKGROUND: Though anatoxin-a (antx-a) is a globally important cyanobacterial neurotoxin in inland waters, information on sublethal toxicological responses of aquatic organisms is limited. We examined influences of (±) antx-a (11-3490 µg/L) on photolocomotor behavioral responses and gene transcription associated with neurotoxicity, oxidative stress and hepatotoxicity, in two of the most common alternative vertebrate and fish models, Danio rerio (zebrafish) and Pimephales promelas (fathead minnow). We selected environmentally relevant treatment levels from probabilistic exposure distributions, employed standardized experimental designs, and analytically verified treatment levels using isotope-dilution liquid chromatography tandem mass spectrometry. Caffeine was examined as a positive control. RESULTS: Caffeine influences on fish behavior responses were similar to previous studies. Following exposure to (±) antx-a, no significant photolocomotor effects were observed during light and dark transitions for either species. Though zebrafish behavioral responses profiles were not significantly affected by (±) antx-a at the environmentally relevant treatment levels examined, fathead minnow stimulatory behavior was significantly reduced in the 145-1960 µg/L treatment levels. In addition, no significant changes in transcription of target genes were observed in zebrafish; however, elavl3 and sod1 were upregulated and gst and cyp3a126 were significantly downregulated in fathead minnows. CONCLUSION: We observed differential influences of (±) antx-a on swimming behavior and gene transcription in two of the most common larval fish models employed for prospective and retrospective assessment of environmental contaminants and water quality conditions. Sublethal responses of fathead minnows were consistently more sensitive than zebrafish to this neurotoxin at the environmentally relevant concentrations examined. Future studies are needed to understand such interspecies differences, the enantioselective toxicity of this compound, molecular initiation events within adverse outcome pathways, and subsequent individual and population risks for this emerging water quality threat.

Sunlight concurrently reduces Prymnesium parvum elicited acute toxicity to fish and prymnesins.

Prymnesium parvum continues to spread globally, producing harmful algal blooms that release toxins known to cause fish kills. While previous work has identified possible P. parvum toxin(s) (e.g., prymnesins, fatty acids, fatty acid amides) and investigated treatment strategies targeted at minimizing cell abundance, studies examining efficacy of treatment approaches to remove toxins are lacking. To understand influences of sunlight on toxins stability and toxicity to fish, acutely toxic P. parvum cultures were exposed to three light scenarios (lab dark control, field dark, and field light) and then evaluated for acute toxicity to fish and prymnesins abundance. Previous work showed acute toxicity to fathead minnow larvae was ameliorated after 2 h of sunlight exposure, and results observed herein found an identical trend. Acute toxicity disappeared in light exposed filtrate, but filtrate exposed to 35 °C without sunlight remained acutely toxic to fish. Additionally, six prymnesins were identified through high-resolution mass spectrometry and abundance corresponded to acute toxicity levels. Prymnesins were present at the highest level in filtrate that was acutely toxic but diminished in filtrate that was exposed to light and correspondingly ameliorated acute toxicity to fish. These findings suggest prymnesins are responsible for measured acute toxicity and are photo-labile, which represents an important implication for treatment strategies.

Toxicology Advances for 21st Century Chemical Pollution.

Pollution represents a leading threat to global health and ecosystems. Systems-based initiatives, including Planetary Health, EcoHealth, and One Health, require theoretical and translational platforms to address chemical pollution. Comparative and predictive toxicology are providing integrative approaches for identifying problematic contaminants, designing less hazardous alternatives, and reducing the impacts of chemical pollution.

Antagonistic Interactions between Benzo[a]pyrene and Fullerene (C60) in Toxicological Response of Marine Mussels.

This study aimed to assess the ecotoxicological effects of the interaction of fullerene (C60) and benzo[a]pyrene (B[a]P) on the marine mussel, Mytilus galloprovincialis. The uptake of nC60, B[a]P and mixtures of nC60 and B[a]P into tissues was confirmed by Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) and Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Biomarkers of DNA damage as well as proteomics analysis were applied to unravel the interactive effect of B[a]P and C60. Antagonistic responses were observed at the genotoxic and proteomic level. Differentially expressed proteins (DEPs) were only identified in the B[a]P single exposure and the B[a]P mixture exposure groups containing 1 mg/L of C60, the majority of which were downregulated (~52%). No DEPs were identified at any of the concentrations of nC60 (p < 0.05, 1% FDR). Using DEPs identified at a threshold of (p < 0.05; B[a]P and B[a]P mixture with nC60), gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that these proteins were enriched with a broad spectrum of biological processes and pathways, including those broadly associated with protein processing, cellular processes and environmental information processing. Among those significantly enriched pathways, the ribosome was consistently the top enriched term irrespective of treatment or concentration and plays an important role as the site of biological protein synthesis and translation. Our results demonstrate the complex multi-modal response to environmental stressors in M. galloprovincialis.

Empirically supported out-of-the-box strategies for science communication by environmental scientists.

Scientists are expected to communicate their research to a wide audience, while often lacking appreciable training. Environmental science poses many value-laden and ethical questions. This necessitates the identification and use of specific strategies or guidelines, which encourage 2-way communication and enable trust in both the experts and the scientific results. The objective of this paper is to give environmental scientists tools for effective science communication based on sound scientific evidence that does not require further specialization in communication studies. Using common scientific search engines in Europe, scientific communication literature that met specific parameters was identified. The summarized data contextualize the importance of science communication in environmental sciences but also highlight the need of scientists for communication experts to aid in establishing objectives for particularly complex topics and audiences. Integr Environ Assess Manag 2019;15:499-504. © 2019 SETAC.

Spheroid Size Does not Impact Metabolism of the ß-blocker Propranolol in 3D Intestinal Fish Model.

Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative in vitro system where individual cell-cell communication and micro-environment more closely represent the in vivo organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish intestine derived RTgutGC cell line. Subsequently, pharmaceutical metabolism (Propranolol), as determined by high performance liquid chromatography, in this intestinal model was examined as a function of spheroid size. Co-efficient of variation between spheroid size was below 12% using the gyratory platform method, with the least variation observed in the highest cell seeding density. The viable, high oxygen micro-environment of the outer rim of the spheroid, as determined by electron paramagnetic resonance (EPR) oximetry, decreased over time, and the hypoxic zone increased as a function of spheroid size. Despite a trend of higher metabolism in smaller spheroids, the formation of micro-environments (quiescent, hypoxic or anoxic) did not significantly affect metabolism or function of an environmentally relevant pharmaceutical in this spheroid model.

Establishment and long-term maintenance of primary intestinal epithelial cells cultured from the rainbow trout, Oncorhynchus mykiss.

A novel method for the establishment and long-term maintenance of ex vivo cultures from intestinal regions of the rainbow trout, Oncorhynchus mykiss (Walbaum), is reported. Adherence of cells was observed within hours, epithelial island formation recorded at 48 h and rapid proliferation with confluence achieved between 9-14 days. In addition to metabolic characterisation, basic morphology of growing cells was characterised using histology, immunofluorescence, transmission electron microscopy (TEM) and transepithelial electrical resistance (TEER). Regional differences in intestinal ethoxyresorufin-O-deethylase (EROD) and 7-ethoxycoumarin-O-deethylation (ECOD) activities in these primary grown enterocytes were compared following exposure to model inducers [i.e. α-NF, ß-NF, B(a)P] which demonstrated significant differences. Regional differences in dietary uptake and metabolism of contaminants can therefore be studied in this in vitro system to increase our understanding of fundamental processes, while concurrently providing a means to reduce the number of fish required for biological studies in line with the principles of the 3Rs (Reduce, Refine and Replace).This article has an associated First Person interview with the first author of the paper.

Assessing the impact of benzo[a]pyrene with the in vitro fish gut model: An integrated approach for eco-genotoxicological studies.

In vitro models are emerging tools for reducing reliance on traditional toxicity tests, especially in areas where information is sparse. For studies of fish, this is especially important for extrahepatic organs, such as the intestine, which, until recently, have been largely overlooked in favour of the liver or gill. Considering the importance of dietary uptake of contaminants, the rainbow trout (Oncorhynchus mykiss) intestine-derived cell line RTgutGC was cultured, to test its suitability as a high-throughput in vitro model. Benzo[a]pyrene (B[a]P) is an important contaminant and a model polycyclic aromatic hydrocarbon (PAH). Over 48 h exposure, a range of endpoints and xenobiotic metabolism rates were examined at three different pH levels indicative of the in vitro (pH 7.5) and in vivo mid-gut (pH 7.7) and hind-gut (pH 7.4) regions as a function of time. These endpoints included (i) cell viability: acid phosphatase (APH) and lactate dehydrogenase (LDH) assays; (ii) glucose uptake; (iii) cytochrome P450 enzyme activity: 7-ethoxyresoorufin-O-deethylase (EROD) assay; (iv) glutathione transferase (GST) activity; (v) genotoxic damage determined using the comet assay. Absence of cell viability loss, in parallel with decrease in the parent compound (B[a]P) in the medium and its subsequent increase in the cells suggested active sequestration, biotransformation, and removal of this representative PAH. With respect to genotoxic response, significant differences were observed at both the sampling times and the two highest concentrations of B[a]P. No significant differences were observed for the different pH conditions. Overall, this in vitro xenobiotic metabolism system appears to be a robust model, providing a basis for further development to evaluate metabolic and toxicological potential of contaminants without use of animals.

Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper.

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the "gut sac" method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner.

Correction: Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

[This corrects the article DOI: 10.1371/journal.pone.0149492.].