A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe.

Transmissible cancers, in which cancer cells themselves act as an infectious agent, have been identified in Tasmanian devils, dogs, and four bivalves. We investigated a disseminated neoplasia affecting geographically distant populations of two species of mussels (Mytilus chilensis in South America and M. edulis in Europe). Sequencing alleles from four loci (two nuclear and two mitochondrial) provided evidence of transmissible cancer in both species. Phylogenetic analysis of cancer-associated alleles and analysis of diagnostic SNPs showed that cancers in both species likely arose in a third species of mussel (M. trossulus), but these cancer cells are independent from the previously identified transmissible cancer in M. trossulus from Canada. Unexpectedly, cancers from M. chilensis and M. edulis are nearly identical, showing that the same cancer lineage affects both. Thus, a single transmissible cancer lineage has crossed into two new host species and has been transferred across the Atlantic and Pacific Oceans and between the Northern and Southern hemispheres.

Hepatic proteome network data in zebrafish (Danio rerio) liver following dieldrin exposure.

Dieldrin is an environmental contaminant that adversely affects aquatic organisms. The data presented in this study are proteomic data collected in liver of zebrafish that were exposed to the pesticide in a dietary exposure. For label free proteomics, data were collected with a quadrupole Time-of-Flight mass spectrometer and for iTRAQ proteomics, data were acquired using a hybrid quadrupole Orbitrap (Q Exactive) MS system. Using formic acid digestion and label free proteomics, 2,061 proteins were identified, and among those, 103 were differentially abundant (p < 0.05 in at least one dose). In addition, iTRAQ proteomics identified 722 proteins in the liver of zebrafish following dieldrin treatment. The label-free approach identified 21 proteins that followed a dose dependent response. Of the differentially abundant proteins identified by iTRAQ, there were 26 unique expression patterns for proteins based on the three doses of dieldrin. Proteins were queried for disease networks to learn more about adverse effects in the liver following dieldrin exposure. Differentially abundant proteins were related to metabolic disease, steatohepatitis and lipid metabolism disorders, drug-induced liver injury, neoplasms, tissue degeneration and liver metastasis. The proteomics data described here is associated with a research article, "Label-free and iTRAQ proteomics analysis in the liver of zebrafish (Danio rerio) following a dietary exposure to the organochlorine pesticide dieldrin" (Simmons et al. 2019). This investigation reveals new biomarkers of toxicity and will be of interest to those studying aquatic toxicology and pesticides.

Label-free and iTRAQ proteomics analysis in the liver of zebrafish (Danio rerio) following dietary exposure to the organochlorine pesticide dieldrin.

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.

Cumulative effects of municipal effluent and parasite infection in yellow perch: A field study using high-throughput RNA-sequencing.

Multiple metabolic, immune and reproductive effects have been reported in fish residing in effluent-impacted sites. Natural stressors such as parasites also have been shown to impact the responses of organisms to chronic exposure to municipal effluent in the St. Lawrence River (Quebec, Canada). In order to comprehensively evaluate the cumulative impacts of anthropogenic and natural stressors on the health of yellow perch, differential mRNA transcription profiles were examined in juvenile females collected from effluent-impacted and upstream sites with low or high infection levels of the larval trematode Apophallus brevis. Transcriptomics was used to identify biological pathways associated with environmental exposure. In total, 3463 isoforms were differentially transcribed between sites. Patterns reflecting the combined effects of stressors were numerically dominant, with a majority of downregulated transcripts (68%). The differentially expressed transcripts were associated with 27 molecular and cellular functions ranging from cellular development to xenobiotic metabolism and were involved in the development and function of 13 organ systems including hematological, hepatic, nervous, reproductive and endocrine systems. Based on RNA-seq results, sixteen genes were measured by qPCR. Significant differences were observed for six genes in fish exposed to both stressors combined, whereas parasites and effluent individually impacted the transcription of one gene. Lysozyme activity, lipid peroxidation, retinol-binding protein and glucose-6-phosphate dehydrogenase were selected as potential biomarkers of effects to study specific pathways of interest. Lipid peroxidation in perch liver was different between sites, parasite loads, and for combined stressors. Overall, results indicated that juvenile yellow perch responded strongly to combined parasite and effluent exposure, suggesting cumulative effects on immune responses, inflammation and lipid metabolism mediated by retinoid receptors. The present study highlight the importance of using a comprehensive approach combining transcriptomics and endpoints measured at higher levels of biological organization to better understand cumulative risks of contaminants and pathogens in aquatic ecosystems.

Halogenated phenolic compounds in wild fish from Canadian Areas of Concern.

Concentrations of halogenated phenolic compounds were measured in the plasma of brown bullhead (Ameiurus nebulosus) from 4 Canadian Areas of Concern (AOCs), to assess exposure to suspected thyroid-disrupting chemicals. Hydroxylated polychlorinated biphenyls (OH-PCBs) were detected in every sample collected in 3 of the AOCs; the detection frequency was lower in samples from the Detroit River AOC. The OH-PCBs most frequently detected were pentachloro, hexachloro, and heptachloro congeners, which are structurally similar to thyroid hormones. Pentachlorophenol (PCP) was detected at highest concentrations (1.8 ng/g) in fish from Prince Edward Bay, the Bay of Quinte Lake reference site, and Hillman Marsh (the Wheatley Harbour reference site), suggesting local sources of contamination. Elevated PCP concentrations were also detected in the plasma of brown bullhead from exposed sites in the Toronto and Region AOC (0.4-0.6 ng/g). Triclosan was consistently detected in the Toronto and Region AOC (0.05-0.9 ng/g), consistent with wastewater emission. Greater concentrations were occasionally detected in the plasma of brown bullhead from the Bay of Quinte AOC. Concentrations of polybrominated diphenyl ethers were highest in the Toronto and Region AOC, and at 2 of the Bay of Quinte AOC exposed sites near Trenton and Belleville. Distribution patterns reflected the properties and usage of the compounds under investigation and the characteristics of each AOC. Environ Toxicol Chem 2017;36:2266-2273. © 2017 SETAC.

A call for collaboration on respectful, person-centered health care in family planning and maternal health.

BACKGROUND: Striking tales of people judged, disrespected, or abused in reproductive, maternal, newborn, child, and adolescent health (RMNCAH) services are commonly exchanged among friends and families throughout the world while remaining sorely under-addressed in global health. Disrespect and abuse of individuals and providers in health services across the RMNCAH continuum must be stopped through collaborative, multi-tiered efforts. CALL FOR COLLABORATION: A new focus on health care quality in the Sustainable Development Goals offers an opportunity to seriously reexamine user experiences and their impact on health care utilization. The new framework provides an opening to redress the insidious problem of negative interactions with care across the RMNCAH services continuum and redraft the blueprint for service delivery and performance measurement, placing individuals and their needs at the center. Both the maternal health and family planning fields are at a turning point in their histories of defining and addressing individuals' experiences of care. In this commentary, we review these histories and the current state-of-the-art in both fields. Though the approaches and language in each sub-field vary, person-centered care principles related to the essential role of individuals' preferences, needs and values, and the importance of informed decision-making, respect, privacy and confidentiality, and non-discrimination, are integral to all. Promoting respectful, person-centered care also requires recognizing the factors that lead to poor treatment of clients, including gender norms and unsupportive working conditions for providers. Lessons can be learned from innovative efforts across the continuum to support health care providers to provide respectful, person-centered care. CONCLUSION: Efforts in the maternal health and family planning fields to define respectful, person-centered care provide a useful foundation from which to connect across the continuum of RMNCAH services. Now is the time to creatively work together to develop new approaches for promoting respectful treatment of individuals in all RMNCAH services.

Inhibition of immune responses and related proteins in Rhamdia quelen exposed to diclofenac.

Nonsteroidal anti-inflammatory drugs are among the most widely detected pharmaceuticals in surface water worldwide. The nonsteroidal anti-inflammatory drug diclofenac is used to treat many types of pain and inflammation. Diclofenac's potential to cause adverse effects in exposed wildlife is a growing concern. To evaluate the effects of waterborne diclofenac on the immune response in Rhamdia quelen (South American catfish), fish were exposed to 3 concentrations of diclofenac (0.2, 2.0, and 20.0 µg/L) for 14 d. Some of the exposed fish were also given an intraperitoneal injection on day 14 of 1 mg/kg of carrageenan to evaluate cell migration to the peritoneum. Total blood leukocyte count and carrageenan-induced leukocyte migration to the peritoneal cavity, particularly of polymorphonuclear cells, were significantly affected for all diclofenac exposure groups. Nitric oxide production was significantly reduced in the diclofenac-treated fish. Plasma and kidney proteins were analyzed by means of liquid chromatography-tandem mass spectrometry in a shotgun proteomic approach. In both plasma and kidney of diclofenac-exposed R. quelen, the expression of 20 proteins related to the inflammatory process, nitric oxide production, leukocyte migration, and the complement cascade was significantly altered. In addition, class I major histocompatibility complex was significantly decreased in plasma of diclofenac-treated fish. Thus, waterborne exposure to diclofenac could lead to suppression of the innate immune system in R. quelen. Environ Toxicol Chem 2017;36:2092-2107. © 2017 SETAC.

Methodology for a mixed-methods multi-country study to assess recognition of and response to maternal and newborn illness.

BACKGROUND: Although maternal and newborn mortality have decreased 44 and 46% respectively between 1990 and 2015, achievement of ambitious Sustainable Development Goal targets requires accelerated progress. Mortality reduction requires a renewed focus on the continuum of maternal and newborn care from the household to the health facility. Although barriers to accessing skilled care are documented for specific contexts, there is a lack of systematic evidence on how women and families identify maternal and newborn illness and make decisions and subsequent care-seeking patterns. The focus of this multi-country study was to identify and describe illness recognition, decision-making, and care-seeking patterns across various contexts among women and newborns who survived and died to ultimately inform programmatic priorities moving forward. METHODS: This study was conducted in seven countries-Ethiopia, Tanzania, Uganda, Nigeria, India, Indonesia, and Nepal. Mixed-methods were utilized including event narratives (group interviews), in-depth interviews (IDIs), focus group discussions (FDGs), rapid facility assessments, and secondary analyses of existing program data. A common protocol and tools were developed in collaboration with study teams and adapted for each site, as needed. Sample size was a minimum of five cases of each type (e.g., perceived postpartum hemorrhage, maternal death, newborn illness, and newborn death) for each study site, with a total of 84 perceived PPH, 45 maternal deaths, 83 newborn illness, 55 newborn deaths, 64 IDIs/FGDs, and 99 health facility assessments across all sites. Analysis included coding within and across cases, identifying broad themes on recognition of illness, decision-making, and patterns of care seeking, and corresponding contextual factors. Technical support was provided throughout the process for capacity building, quality assurance, and consistency across sites. CONCLUSION: This study provides rigorous evidence on how women and families recognize and respond to maternal and newborn illness. By using a common methodology and tools, findings not only were site-specific but also allow for comparison across contexts.

Widespread transmission of independent cancer lineages within multiple bivalve species.

Most cancers arise from oncogenic changes in the genomes of somatic cells, and while the cells may migrate by metastasis, they remain within that single individual. Natural transmission of cancer cells from one individual to another has been observed in two distinct cases in mammals (Tasmanian devils and dogs), but these are generally considered to be rare exceptions in nature. The discovery of transmissible cancer in soft-shell clams (Mya arenaria) suggested that this phenomenon might be more widespread. Here we analyse disseminated neoplasia in mussels (Mytilus trossulus), cockles (Cerastoderma edule), and golden carpet shell clams (Polititapes aureus) and find that neoplasias in all three species are attributable to independent transmissible cancer lineages. In mussels and cockles, the cancer lineages are derived from their respective host species; however, unexpectedly, cancer cells in P. aureus are all derived from Venerupis corrugata, a different species living in the same geographical area. No cases of disseminated neoplasia have thus far been found in V. corrugata from the same region. These findings show that transmission of cancer cells in the marine environment is common in multiple species, that it has originated many times, and that while most transmissible cancers are found spreading within the species of origin, cross-species transmission of cancer cells can occur.

Evaluating the toxic potential of benzothiazoles with the rainbow trout cell lines, RTgill-W1 and RTL-W1.

Benzothiazole (BTHs) are environmental contaminants of emerging concern for which little toxicological information is available. Therefore the toxic potential of twelve BTHs was evaluated with two rainbow trout epithelial cell lines, RTgill-W1 and RTL-W1. The BTHs were benzothiazole (BTH), 3,3'-diethylthia dicarbocyanine iodide (DTDC), C.I. sulphur orange 1 (SO), 2-mercaptobenzothiazole (2MBTH), zinc 2-mercaptobenzothiazole (ZnMBTH), sodium 2-mercaptobenzothiazole (NaMBTH), 2-hydroxy-benzothiazole (OHBTH), 2- aminobenzothiazole (2ABTH), C.I. vat yellow 2 (VY), N,N-dicyclohexyl-2-benzothiazolsulfene amide (NNA), 2,2'-dithiobis (benzothiazole) (DBTH) and 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid (MBTHS). All BTHs, except for NNA, DBTH, and MBTHS, caused both cytotoxicity and a transitory elevation in reactive oxygen species (ROS) levels. Yet, neither N-acetyl cysteine (NAC) nor IM-54 inhibited cytotoxicity, suggesting that ROS imbalance did not contribute to cell death. Cell death was not blocked by Necrostatin-1 nor accompanied by DNA laddering, suggesting that neither necroptosis nor apoptosis took place. The comet assay revealed DNA strand breaks after exposures to 2ABTH and OHBTH for 1 day and to BTH for 12 days. In RTL-W1, cytochrome P4501A was induced noticeably by 2ABTH, OHBTH, and MBTHS and weakly by NaMBTH, ZnMBTH, SO, VY, and NNA, suggesting that these BTHs have the potential to alter xenobiotic metabolism and activate the aryl hydrocarbon receptor. In summary, several toxic actions were initiated in vitro by some but not all BTHs, warranting further study of these BTHs in vivo.

Plasma proteome profiles of White Sucker (Catostomus commersonii) from the Athabasca River within the oil sands deposit.

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.

Use of the rainbow trout cell lines, RTgill-W1 and RTL-W1 to evaluate the toxic potential of benzotriazoles.

Epithelial cell lines, RTgill-W1 and RTL-W1 from respectively gill and liver of rainbow trout, Onchorhynchus mykiss (Walbaum), were used to evaluate the toxic potential of six benzotriazoles (BTRs) and tolytriazole (TT), which is a commercial mixture of 4-methyl-1H-benzotriazole (4MBTR) and 5-methyl-1H-benzotriazole (5MBTR). The other BTRs were 1H-benzotriazole (1H-BTR), 5-chlorobenzotriazole (5CBTR), 1-hydroxybenzotriazole (1OHBTR) and 5,6-dimethyl-1H-benzotriazole monohydrate (DM). Except for DM, all BTRs were cytotoxic at concentrations above 15mg/L and transitorily elevated reactive oxygen species (ROS) levels. Neither N-acetyl cysteine (NAC) nor IM-54 inhibited cytotoxicity, suggesting that ROS were not the major cause of the cell death. Cell death was not blocked by Necrostatin nor accompanied by DNA laddering, suggesting that the cell death mechanism was neither necroptosis nor apoptosis. As judged by the comet assay, DNA strand breaks were detected with three BTRs: 4MBTR, 5MBTR and 5CBTR. In RTL-W1, the BTRs weakly induced cytochrome P4501A, suggesting that they have the potential to alter xenobiotic metabolism and activate the aryl hydrocarbon receptor. In summary, the toxic potential of BTRs appears to be limited to only high concentrations, which are higher than have been measured in the environment to date.

The p53 inhibitor, pifithrin-α, disrupts microtubule organization, arrests growth, and induces polyploidy in the rainbow trout gill cell line, RTgill-W1.

Pifithrin-α (PFT-α) blocks p53-dependent transcription and is an example of the many drugs being developed to target the p53 pathway in humans that could be released into the environment with potential impacts on aquatic animals if they were to become successful pharmaceuticals. In order to understand how p53 drugs might act on fish, the effects of PFT-α on rainbow trout gill epithelial cell line, RTgill-W1, were studied. PFT-α was not cytotoxic to RTgill-W1 in cultures with or without fetal bovine serum (FBS), but at 5.25µg/ml, PFT-α completely arrested proliferation. When FBS was present, PFT-α increased the number of polyploid cells over 12days. Those results suggest that like in mammals, p53 appears to regulate ploidy in fish. However, several effects were seen that have not been observed with mammalian cells. PFT-α caused a transient rise in the mitotic index and a disruption in cytoskeletal microtubules. These results suggest that in fish cells PFT-α affects microtubules either directly through an off-target action on tubulin or indirectly through an on-target action on p53-regulated transcription.

Omics for aquatic ecotoxicology: control of extraneous variability to enhance the analysis of environmental effects.

There are multiple sources of biological and technical variation in a typical ecotoxicology study that may not be revealed by traditional endpoints but that become apparent in an omics dataset. As researchers increasingly apply omics technologies to environmental studies, it will be necessary to understand and control the main source(s) of variability to facilitate meaningful interpretation of such data. For instance, can variability in omics studies be addressed by changing the approach to study design and data analysis? Are there statistical methods that can be employed to correctly interpret omics data and make use of unattributed, inherent variability? The present study presents a review of experimental design and statistical considerations applicable to the use of omics methods in systems toxicology studies. In addition to highlighting potential sources that contribute to experimental variability, this review suggests strategies with which to reduce and/or control such variability so as to improve reliability, reproducibility, and ultimately the application of omics data for systems toxicology.

Horizontal transmission of clonal cancer cells causes leukemia in soft-shell clams.

Outbreaks of fatal leukemia-like cancers of marine bivalves throughout the world have led to massive population loss. The cause of the disease is unknown. We recently identified a retrotransposon, Steamer, that is highly expressed and amplified to high copy number in neoplastic cells of soft-shell clams (Mya arenaria). Through analysis of Steamer integration sites, mitochondrial DNA single-nucleotide polymorphisms (SNPs), and polymorphic microsatellite alleles, we show that the genotypes of neoplastic cells do not match those of the host animal. Instead, neoplastic cells from dispersed locations in New York, Maine, and Prince Edward Island (PEI), Canada, all have nearly identical genotypes that differ from those of the host. These results indicate that the cancer is spreading between animals in the marine environment as a clonal transmissible cell derived from a single original clam. Our findings suggest that horizontal transmission of cancer cells is more widespread in nature than previously supposed.

Lithium an emerging contaminant: bioavailability, effects on protein expression, and homeostasis disruption in short-term exposure of rainbow trout.

Worldwide production of lithium (Li) has increased dramatically during the past decade, driven by the demand for high charge density batteries. Information about Li in the aquatic environment is limited. The present study was designed to explore the effects of Li in rainbow trout (Oncorhynchus mykiss). Juvenile trout were exposed to a nominal concentration of 1.0mg Li/L in three separate exposures. Major ion concentrations were measured in brain and plasma by ion chromatography. Plasma proteins and fatty acids were measured by HPLC-MS/MS. Lithium accumulated in the brain and plasma. Arachidonic acid was elevated in plasma after 48h. Elevated concentrations of Li in brain were associated with depressed concentrations of sodium, magnesium, potassium and ammonium relative to the control. In plasma, sodium and calcium were also depressed. Several changes occurred to plasma proteins corresponding to Li exposure: inhibition of prostaglandin synthase (Ptgs2), increased expression of copper transporting ATP synthases, and Na(+)/K(+) ATPase. To our knowledge, ours is the first study to demonstrate elevated Li concentrations in fish brain, with associated effects on ion regulation.

Activation of transcription and retrotransposition of a novel retroelement, Steamer, in neoplastic hemocytes of the mollusk Mya arenaria.

Bivalve mollusks of the North Atlantic, most prominently the soft shell clam Mya arenaria, are afflicted with an epidemic transmissible disease of the circulatory system closely resembling leukemia. The disease is characterized by a dramatic expansion of blast-like cells in the hemolymph with high mitotic index. Examination of hemolymph of diseased clams revealed high levels of reverse transcriptase activity, the hallmark of retroviruses and retroelements. By deep sequencing of RNAs from hemolymph, we identified transcripts of a novel retroelement, here named Steamer. The DNA of the element is marked by long terminal repeats and encodes a single large protein with similarity to mammalian retroviral Gag-Pol proteins. Steamer mRNA levels were specifically elevated in diseased hemocytes, and high expression was correlated with disease status. DNA copy number per genome was present at enormously high levels in diseased hemocytes, indicative of extensive reverse transcription and retrotransposition. Steamer activation in M. arenaria is an example of a catastrophic induction of genetic instability that may initiate or advance the course of leukemia.

The p53/HSP70 inhibitor, 2-phenylethynesulfonamide, causes oxidative stress, unfolded protein response and apoptosis in rainbow trout cells.

The effect of 2-phenylethynesulfonamide (PES), which is a p53 and HSP70 inhibitor in mammalian cells, was studied on the rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1, in order to evaluate PES as a tool for understanding the cellular survival pathways operating in fish. As judged by three viability assays, fish cells were killed by 24h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspases activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to accumulate in the detergent-insoluble fraction, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest. PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways.

Estrogen-like effects in male goldfish co-exposed to fluoxetine and 17 alpha-ethinylestradiol.

The antidepressant fluoxetine (FLX) and the synthetic estrogen, 17 alpha-ethinylestradiol (EE2), are present in municipal sewage discharges. To better understand possible interactions between them, male goldfish were exposed to an ethanol control or to nominal concentrations of FLX (0.54 µg/L) and EE2 (5 ng/L) alone and in combination for 14 days. Real-time reverse-transcription polymerase chain reaction was used to assess effects on hepatic gene expression and liquid chromatography tandem mass spectrometry to analyze the plasma proteome. The results showed an increase in estrogen receptor alpha (esr1) and vitellogenin (vtg) gene expression by 1.9-2.4-fold in the FLX and EE2 groups, but this did not reach statistical significance. In contrast, co-exposure up regulated esr1 and vtg gene expression by 5.5- and 5.3-fold, respectively. Fluoxetine and EE2 alone did not affect estrogen receptor beta (esr2), but the co-exposure down regulated esr2 expression by 50%. There was a significant increase in the number of plasma proteins that were related to endocrine system disorders in the FLX and FLX plus EE2 groups. The level of VTG protein was increased in the plasma from goldfish exposed to EE2, FLX, and FLX plus EE2. Our study demonstrates that low concentrations of FLX and EE2 in a simple mixture produce strong estrogen-like effects in the male goldfish.

The rad1 gene in Rainbow Trout (Oncorhynchus mykiss) is highly conserved and may express proteins from non-canonical spliced isoforms.

Cell-cycle checkpoint proteins maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. RAD1 is a checkpoint protein involved in the sensing of damaged DNA and is a part of the 9-1-1 complex. In this project rainbow trout rad1 (rtrad1) was cloned, sequenced, expressed as a recombinant protein and anti-rtRAD1 antibodies were developed. RAD1 protein levels were characterized in various rainbow trout tissues. It was determined that an 840 bp open-reading frame encodes 279 aa with a predicted protein size of 31 kDa. The rtRAD1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify three non-canonical splice variants of rtrad1, two of which are capable of forming functional proteins. The rad1 splice variant that encodes an 18 kDa protein appears to be abundant in rainbow trout spleen, heart and gill tissue and in the RTgill-W1 cell-line. Based on the genomic rtrad1 sequence the splice variants contain only partial exons which are consistent with the splicing of rad1 variants in mammals. This is the first time that rad1 has been fully characterized in a fish species.