Hepatic Transcriptomic Responses to Ethinylestradiol in Embryonic Japanese Quail and Double-Crested Cormorant.

Understanding species differences in sensitivity to toxicants is a critical issue in ecotoxicology. We recently established that double-crested cormorant (DCCO) embryos are more sensitive than Japanese quail (JQ) to the developmental effects of ethinylestradiol (EE2). We explored how this difference in sensitivity between species is reflected at a transcriptomic level. The EE2 was dissolved in dimethyl sulfoxide and injected into the air cell of eggs prior to incubation at nominal concentrations of 0, 3.33, and 33.3 µg/g egg weight. At midincubation (JQ 9 days; DCCO 16 days), livers were collected from five embryos/treatment group for RNA sequencing. Data were processed and analyzed using EcoOmicsAnalyst and ExpressAnalyst. The EE2 exposure dysregulated 238 and 1,987 genes in JQ and DCCO, respectively, with 78 genes in common between the two species. These included classic biomarkers of estrogen exposure such as vitellogenin and apovitellenin. We also report DCCO-specific dysregulation of Phase I/II enzyme-coding genes and species-specific transcriptional ontogeny of vitellogenin-2. Twelve Kyoto Encyclopedia of Genes and Genomes pathways and two EcoToxModules were dysregulated in common in both species including the peroxisome proliferator-activated receptor (PPAR) signaling pathway and fatty acid metabolism. Similar to previously reported differences at the organismal level, DCCO were more responsive to EE2 exposure than JQ at the gene expression level. Our description of differences in transcriptional responses to EE2 in early life stage birds may contribute to a better understanding of the molecular basis for species differences. Environ Toxicol Chem 2024;43:772-783. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Comparing Transcriptomic Responses to Chemicals Across Six Species Using the EcoToxChip RNASeq Database.

The EcoToxChip project includes RNA-sequencing data from experiments involving model (Japanese quail, fathead minnow, African clawed frog) and ecological (double-crested cormorant, rainbow trout, northern leopard frog) species at multiple life stages (whole embryo and adult) exposed to eight chemicals of environmental concern known to perturb a wide range of biological systems (ethinyl estradiol, hexabromocyclododecane, lead, selenomethionine, 17ß trenbolone, chlorpyrifos, fluoxetine, and benzo[a]pyrene). The objectives of this short communication were to (1) present and make available this RNA-sequencing database (i.e., 724 samples from 49 experiments) under the FAIR principles (FAIR data are data which meet principles of findability, accessibility, interoperability, and reusability), while also summarizing key meta-data attributes and (2) use ExpressAnalyst (including the Seq2Fun algorithm and EcoOmicsDB) to perform a comparative transcriptomics analysis of this database focusing on baseline and differential transcriptomic changes across species-life stage-chemical combinations. The database is available in NCBI GEO under accession number GSE239776. Across all species, the number of raw reads per sample ranged between 13 and 58 million, with 30% to 79% of clean reads mapped to the "vertebrate" subgroup database in EcoOmicsDB. Principal component analyses of the reads illustrated separation across the three taxonomic groups as well as some between tissue types. The most common differentially expressed gene was CYP1A1 followed by CTSE, FAM20CL, MYC, ST1S3, RIPK4, VTG1, and VIT2. The most common enriched pathways were metabolic pathways, biosynthesis of cofactors and biosynthesis of secondary metabolites, and chemical carcinogenesis, drug metabolism, and metabolism of xenobiotics by cytochrome P450. The RNA-sequencing database in the present study may be used by the research community for multiple purposes, including, for example, cross-species investigations, in-depth analyses of a particular test compound, and transcriptomic meta-analyses. Environ Toxicol Chem 2024;00:1-6. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Cytotoxicity of 19 Pesticides in Rainbow Trout Gill, Liver, and Intestinal Cell Lines.

The rainbow trout gill cell line (RTgill-W1), via test guideline 249 of the Organisation for Economic Co-operation and Development, has been established as a promising New Approach Methodology, although to advance confidence in the method more case studies are needed that: 1) expand our understanding of applicability domains (chemicals with diverse properties); 2) increase methodological throughput (96-well format); and 3) demonstrate biological relevance (in vitro to in vivo comparisons; gill vs. other cells). Accordingly, the objective of our study was to characterize the cytotoxicity of 19 pesticides against RTgill-W1 cells, and also liver (RTL-W1) and gut epithelial (RTgutGC) cell lines, and then to compare the in vitro and in vivo data. Of the 19 pesticides tested, 11, 9, and 8 were cytotoxic to the RTgill-W1, RTL-W1, and RTgutGC cells, respectively. Six pesticides (carbaryl, chlorothalonil, chlorpyrifos, dimethenamid-P, metolachlor, and S-metolachlor) were cytotoxic to all three cell lines. Aminomethylphosphonic acid, chlorantraniliprole, dicamba, diquat, imazethapyr, and permethrin exhibited cell-line-specific toxicity. No cytotoxic responses were observed for three herbicides (atrazine, glyphosate, and metribuzin) and four insecticides (clothianidin, diazinon, imidacloprid, and thiamethoxam). When cytotoxicity was measured, there was a strong correlation (rs = 0.9, p < 0.0001) between in vitro median effect concentration (EC50) values (based on predicted concentrations using the In Vitro Mass Balance Model Equilibrium Partitioning (IV-MBM EQP) Ver. 2.1) derived from RTgill-W1 and RTL-W1 cells with in vivo median lethal concentration (LC50) values from 96-h acute toxicity studies with trout. In all 28 cases, the in vitro EC50 was within 18-fold of the in vivo LC50. These data help increase our understanding of the ecotoxicological domains of applicability for in vitro studies using cultured rainbow trout cells, while also demonstrating that these assays performed well in a 96-well format and have promise to yield data of biological relevance. Environ Toxicol Chem 2024;00:1-13. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Cumulative effects of cadmium and natural stressors (temperature and parasite infection) on molecular and biochemical responses of juvenile rainbow trout.

The simultaneous presence of natural and anthropogenic stressors in aquatic ecosystems can challenge the identification of factors causing decline in fish populations. These stressors include chemical mixtures and natural abiotic and biotic factors such as water temperature and parasitism. Effects of cumulative stressors may vary from antagonism to synergism at the organismal or population levels and may not be predicted from exposure to individual stressors. This study aimed to evaluate the combined effects of chronic exposure to cadmium (Cd) and elevated water temperature (23 °C) or parasite infection in juvenile rainbow trout (Oncorhynchus mykiss) using a multi-level biological approach, including RNA-sequencing. Fish were exposed to diet-borne Cd (6 µg Cd/g wet feed), individually and in combination with thermal (23 °C) or parasitic stressors, for 28 days. The parasite challenge consisted of a single exposure to glochidia (larvae) of the freshwater mussel (Strophitus undulatus), which encysts in fish gills, fins and skin. Results indicated lower fish length, weight, and relative growth rate in fish exposed to a higher water temperature (23 °C). Body condition and hepatosomatic index of trout were, however, higher in the 23 °C temperature treatment compared to the control fish kept at 15 °C. Exposure to thermal stress or parasitism did not influence tissue Cd bioaccumulation. More than 700 genes were differentially transcribed in fish exposed to the individual thermal stress treatment. However, neither Cd exposure nor parasite infection affected the number of differentially transcribed genes, compared to controls. The highest number of differentially transcribed genes (969 genes) was observed in trout exposed to combined Cd and high temperature stressors; these genes were mainly related to stress response, protein folding, calcium metabolism, bone growth, energy metabolism, and immune system; functions overlapped with responses found in fish solely exposed to higher water temperature. Only 40 genes were differentially transcribed when fish were exposed to Cd and glochidia and were related to the immune system, apoptosis process, energy metabolism and malignant tumor. These results suggest that dietary Cd may exacerbate the temperature stress and, to a lesser extent, parasitic infection stress on trout transcriptomic responses. Changes in the concentrations of liver ethoxyresorufin-o-deethylase, heat shock protein 70 and thiobarbituric acid reactive substances coupled to changes in the activities of cellular glutathione S-transferase and glucose-6-phosphate dehydrogenase were also observed at the cellular level. This study may help understand effects of freshwater fish exposure to cumulative stressors in a changing environment.

Treatment of patients with primary retroperitoneal sarcoma: predictors of outcome from an Australian specialist sarcoma centre.

BACKGROUND: Several unanswered questions surround the management of retroperitoneal sarcoma (RPS). Guidelines recommend treatment by a multidisciplinary team at a specialized referral centre. The objective of this study was to describe the management of RPS at an Australian specialist sarcoma centre, comparing outcomes to international standards and analysing for predictors of local failure. METHODS: A retrospective review of a prospectively maintained database was performed on patients with RPS treated between 2008 and 2016. A 5-year outcome analyses focussed on patients undergoing curative-intent surgery for primary, non-metastatic RPS. RESULTS: Eighty-eight patients underwent surgery for primary RPS. Five-year overall survival was 66%, 5-year freedom from local recurrence was 65% and 5-year freedom from distant metastasis was 71%. Overall survival was associated with tumour grade (hazard ratio (HR) 6.1, P < 0.001) and histologic organ invasion (HR 5.7, P < 0.001). Variables associated with improved freedom from local recurrence were macroscopically complete resection (HR 0.14, P < 0.001) and neoadjuvant radiotherapy (HR 0.33, P = 0.014). Treatment at a specialist sarcoma centre was associated with a higher rate of preoperative biopsy and neoadjuvant radiotherapy (both with P < 0.001). There was a trend towards improved local control for patients undergoing surgery at a specialist centre (P = 0.055). CONCLUSION: This is the largest Australian series of RPS and outcomes are comparable to major international sarcoma centres. Patients treated at a specialist centre had higher rates of preoperative diagnosis and tailored therapy which was associated with improved outcomes. Patients with suspected RPS should be referred to a specialist centre for optimal preoperative evaluation and multidisciplinary management.