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Antineoplastic medications are present in aquatic environments and are measured at relatively high concentrations in hospital sewage effluent. Thus, it is important to characterize risk associated with waterborne exposures to anticancer drugs. The drug 5-fluorouracil (5-FU) is used to treat several types of cancers, acting to inhibit cell division and cellular metabolism. The objectives of this study were to determine the effects of 5-FU on developmental endpoints and lipid composition in zebrafish. 5-FU did not negatively affect development nor survival in developing zebrafish at concentrations up to 1000 μg/L. However, 5-FU increased neutral lipid content in zebrafish larvae, indicating potential for lipid dysregulation. To further discern effects on lipids, lipidomics was conducted and a total of 164 lipids belonging to 14 lipid classes were identified. Significant changes (false discovery rate < 0.05) in abundance were detected for 19 lipids including some ceramides, ether-linked phosphatidylethanolamines, and sphingomyelins among others. We also measured the expression levels of 14 lipid-related enzymes and transporters (e.g., acox3, dgat1, fads2, fasn, elovl2) using real-time PCR; however, mRNA abundance levels were not affected, suggesting transcriptional changes may not be a primary mechanism underlying lipid dysregulation. Locomotor activity was measured in zebrafish as lipids are needed for swimming activity in larvae. Exposure to 5-FU did not affect locomotor activity up to 1000 μg/L. We conclude that lipids accumulate in larval zebrafish with exposure to 5-FU, which can subsequently affect lipid composition. These data reveal potential lipid signatures of 5-FU exposure and contribute to risk assessments for antineoplastic exposure in aquatic environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

PFAAs (Perfluoroalkyl acids) are a class of bioaccumulative, persistent and ubiquitous environmental contaminants which primarily occupy the hydrosphere and its sediments. Currently, a paucity of toxicological information exists for short chain PFAAs and complex mixtures. In order to address these knowledge gaps, we performed a 3-week, aqueous exposure of rainbow trout to 3 different concentrations of a PFAA mixture (50, 100 and 500 ng/L) modeled after the composition determined in Lake Ontario. We conducted an additional set of exposures to individual PFAAs (25 nM each of PFOS (12,500 ng/L), PFOA (10,300 ng/L), PFBS (7500 ng/L) or PFBA (5300 ng/L) to evaluate differences in biological response across PFAA congeners. Untargeted proteomics and phosphorylated metabolomics were conducted on the blood plasma and head kidney tissue to evaluate biological response. Plasma proteomic responses to the mixtures revealed several unexpected outcomes including Similar proteomic profiles and biological processes as the PFOS exposure regime while being orders of magnitude lower in concentration and an atypical dose response in terms of the number of significantly altered proteins (FDR < 0.1). Biological pathway analysis revealed the low mixture, medium mixture and PFOS to significantly alter (FDR < 0.05) a number of processes including those involved in lipid metabolism, oxidative stress and the nervous system. We implicate plasma increases in PPARD and PPARG as being directly related to these biological processes as they are known to be important regulators in all 3 processes. In contrast to the blood plasma, the high mixture and PFOA exposure regimes caused the greatest change to the head kidney proteome, altering many proteins being involved in lipid metabolism, oxidative stress and inflammation. Our findings support the pleiotropic effect PFAAs have on aquatic organisms at environmentally relevant doses including those on PPAR signaling, metabolic dysregulation, immunotoxicity and neurotoxicity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Denina Simmons reports financial support and administrative support were provided by Ontario Tech University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Protein expression patterns adapt to various cues to meet the needs of an organism. The dynamicity of an organism's proteome can therefore reveal information about an organism's health. Proteome databases contain limited information regarding organisms outside of medicinal biology. The UniProt human and mouse proteomes are extensively reviewed and ∼50 % of both proteomes include tissue specificity, while >99 % of the rainbow trout proteome lacks tissue specificity. This study aimed to expand knowledge on the rainbow trout proteome with a focus on understanding the origin of blood plasma proteins. Blood, brain, heart, liver, kidney, and gills were collected from adult rainbow trout, plasma and tissue proteins were analyzed using liquid chromatography tandem mass spectrometry. Over 10,000 proteins were identified across all groups. Our data indicated that the majority of the plasma proteome is shared amongst multiple tissue types, though 4-7 % of the plasma proteome is uniquely originated from each tissue (gill > heart > liver > kidney > brain)., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Denina Simmons reports financial support was provided by Natural Sciences and Engineering Research Council of Canada., (Copyright © 2023. Published by Elsevier Inc.)

The incidence of hypoxia in water bodies is increasing more rapidly than aquatic life can adapt. This study aimed to determine the effects of hypoxia on fish physiology, as well as protein expression through proteomics. To do this, 40 rainbow trout were divided into normoxic control (11.5 mg/L dissolved oxygen) and hypoxic treatment (5 mg/L dissolved oxygen) tanks for a period of 7 days. Fish were then anesthetized and blood was sampled. Fish were then euthanized and heart and liver samples were taken. Blood glucose, cortisol and lipid, body and liver mass, fork length, hematocrit and, blood cell counts and global heart methylation were measured. Red blood cell counts were significantly lower, while hematocrit and mean corpuscular volume were significantly higher in the hypoxic treatment. Global DNA methylation was significantly decreased in hypoxic heart tissue. Plasma cortisol and 18:1 monoacylglyerol increased, while 15:0-18:1 phosphatidylethanolamine, and 18:1 lysophosphatidylethanolamine decreased in plasma of rainbow trout under hypoxic conditions. Plasma proteomics revealed 70 significantly altered proteins (p < 0.05) in the hypoxia treatment (Data are available via ProteomeXchange with identifier PXD026589). Many of these molecular changes appear to be related to the observed increase in red blood cell volume and epigenetic modifications, as well as to angiogenesis, lipid, and glucose metabolism. This study highlights a range of cellular and molecular responses in the blood and plasma of freshwater fish that may be phenotypic adaptions to hypoxia, and that could aid in diagnosing the health status of wild fish populations using several, potential, discovered biomarkers., (Copyright © 2021. Published by Elsevier Inc.)

Proteomics plays a significant role in discerning the effects of chemical exposures in animal taxa. Multi-omics applications have become more pervasive in toxicology, however questions remain about whether proteomics is being utilized by the community to its full potential - are we placing too much stock in transcriptomics and other omics approaches for developing adverse outcome pathways? Proteins are more relevant than transcripts because they are direct mediators of the resulting phenotype. There is also rarely perfect stoichiometry between transcript and protein abundance and transcript abundance may not accurately predict physiologic response. Proteins direct all levels of phenotype: structural proteins dictate physical form, enzymes catalyze biochemical reactions, and proteins act as signaling proteins, antibodies, transporters, ion pumps, and transcription factors to control gene expression. Molecular initiating events (MIEs) of AOPs predominantly occur at the level of the protein (e.g. ligand-receptor binding) and proteomics can elucidate novel MIEs and mapping KEs in AOPs. This critical review highlights the need for proteomics in multi-omics studies in environmental toxicology and outlines steps required for inclusion and wider acceptance in chemical risk assessment. We also present case studies of multi-omics approaches that utilize proteomics and discuss some of the challenges and opportunities for proteomics in comparative ecotoxicology. Our intention is not to minimize the importance of other omics technologies, as each has strengths and limitations, but rather to encourage researchers to consider proteomics-based methods in multi-omics studies and AOP development., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The objectives of this study were to assess the lethal and sub-lethal effects of the aquatic herbicide commercial formulation, Reward® (373 g/L DB), using application scenarios prescribed by the manufacturer. Specifically, a 14 d period between applications of Reward® in a water body undergoing treatment is required, yet the effects of these 'pulse' exposure scenarios on aquatic wildlife such as fish are unknown. In the first experiment early life stage FHM were exposed to a continuous DB concentrations from 0.105-12.6 mg/L which yielded a larval 7 d LC 50 of 2.04 mg/L as well as a significant decrease in body mass (25.0 ± 11.6%) at the 1.18 mg/L Reward® concentration. In a second experiment, FHM larvae were exposed for 24 h and then reared in clean water for 14 d followed by a second 24 h exposure to Reward®. The 16 d LC 50 value was 4.19 mg/L. In a third experiment, adult FHM were exposed in a pulse/discontinuous manner to Reward® with a calculated 21 d LC 50 value of 6.71 mg/L. No significant changes in gonadosomatic index or fecundity of the F 1 generation's hatch success were found when eggs from exposed adults were then reared in clean water. Proteome analyses of whole FHM larvae from the discontinuous/pulse exposure showed the primary gene ontology molecular functions of the proteins in fish exposed to 3.78 mg/L DB that resulted in ~30% mortality with positive or negative differential abundance (p-value < .2) were: structural molecule activity; identical protein binding; structural constituent of cytoskeleton; ion binding; calcium ion binding; cytoskeletal protein binding; actin binding; and, ATP binding. These findings suggest that concentrations causing adverse effects occur above the maximum concentration predicted by the manufacturer when applied according to the label (i.e. >0.37 mg/L)., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019. Published by Elsevier Inc.)

Microcystins that are cell-bound within Microcystis have demonstrated the ability to cause lethal and reproductive impairment in Daphnia, who constitute an important part of aquatic food chains and are known to feed on viable cyanobacterial cells. Recent advances in environmental toxicogenomics can be used to better understand the mechanistic effects from exposure to cell-bound microcystins in Daphnia; however, there remains a need to examine the effects of microcystins exposure as a function of dose and time in order to help elucidate the progression of (sub-)lethal effects. This study examines the effects of cell-bound microcystin exposure in Daphnia magna as a function of dose and time with shotgun proteomics in order to measure and provide insightful evidence describing functional mechanisms from, and relationships between, protein populations in response to toxic Microcystis aeruginosa. We further characterize the life-history fitness of D. magna in the presence of toxic exposure by measuring somatic growth rate. Chronic dietary exposure to cell-bound microcystins reduced the somatic growth rate of D. magna. Through proteomics analysis, we identified a significant increase in abundance of proteins related to reproductive success and development, removal of superoxide radicals, and motor activity in D. magna parents exposed to cell-bound microcystins at sub-lethal concentrations. We also identified a significant decrease in abundance of proteins related to apoptosis, metabolism, DNA damage repair, and immunity in D. magna neonates. This information will improve our understanding of the risks posed by cell-bound microcystins to cladocerans in freshwater ecosystems., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The organochlorine dieldrin (DLD) bioaccumulates in lipid-rich tissues and is associated with immunosuppression, altered metabolism, and cancer. The objective of this study was to determine the effect of DLD on the hepatic proteome in zebrafish following dietary treatment as the liver is central to metabolism. Females were fed a control dose or one of three doses of DLD-contaminated food pellets over 21 days. Both label-free and iTRAQ proteomics were conducted as two complementary methods to expand coverage of the proteome. Label-free proteomics quantified 1563 proteins: 6 proteins showed a linear dose-response with DLD. iTRAQ quantified >3500 proteins; 5 proteins were decreased and 34 proteins were increased in abundance within the liver with all three doses. Overall, DLD reduced the abundance of proteins associated with glucose and cholesterol metabolism, lipid oxidation, liver function, and immune-related processes. Few proteins were identified by both methods as being altered (~1%), suggesting that each method detected different subsets of proteins. Protein responses in the liver were largely dependent on dose, however proteins related to liver and organ function, centrosome separation, glucose/energy metabolism, and immune-related pathways were confirmed by each independent technique and were suppressed with DLD exposure. This study identifies proteomic responses that are associated with organochlorine-induced hepatotoxicity. BIOLOGICAL SIGNIFICANCE: Environmental contaminants cause hepatotoxicity because the liver is the major organ for detoxification. The legacy pesticide dieldrin significantly bioaccumulates in tissues, and can affect molecular processes that can lead to liver pathology. LC MS/MS proteomics identified protein networks related to tumors, energy homeostasis, and chromosomal separation as those affected by dietary exposure to dieldrin. We applied two orthogonal mass spectrometry-based methods to more completely survey the liver proteome, strengthening data interpretation. These data improve understanding as to the effects of organochlorine pesticide toxicity in the liver and the study identifies proteome networks that can contribute to adverse outcome pathways for pesticide exposure., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There are questions about the potential for oil sands related chemicals to enter the Athabasca River, whether from tailing ponds, atmospheric deposition, precipitation, or transport of mining dust, at concentrations sufficient to negatively impact the health of biota. We applied shotgun proteomics to generate protein profiles of mature male and female White Sucker (Catostomus commersonii) that were collected from various sites along the main stem of the Athabasca River in 2011 and 2012. On average, 399±131 (standard deviation) proteins were identified in fish plasma from each location in both years. Ingenuity Pathway Analysis software was used to determine the proteins' core functions and to compare the datasets by location, year, and sex. Principal component analysis (PCA) was used to determine if variation in the number of proteins related to a core function among all male and female individuals from both sampling years was affected by location. The core biological functions of plasma proteins that were common to both sampling years for males and females from each location were also estimated separately (based on Ingenuity's Knowledge Base). PCA revealed site-specific differences in the functional characteristics of the plasma proteome from white sucker sampled from downstream of oil sands extraction facilities compared with fish from upstream. Plasma proteins that were unique to fish downstream of oil sands extraction were related to lipid metabolism, small molecule biochemistry, vitamin and mineral metabolism, endocrine system disorders, skeletal and muscular development and function, neoplasia, carcinomas, and gastrointestinal disease., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)