B cell expression of E3 ubiquitin ligase Cul4b promotes chronic gammaherpesvirus infection in vivo.

IMPORTANCE: The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologic agents of numerous B cell lymphomas. A hallmark of gammaherpesvirus infection is their ability to establish lifelong latency in B cells. However, the specific mechanisms that mediate chronic infection in B cells in vivo remain elusive. Cellular E3 ubiquitin ligases regulate numerous biological processes by catalyzing ubiquitylation and modifying protein location, function, or half-life. Many viruses hijack host ubiquitin ligases to evade antiviral host defense and promote viral fitness. Here, we used the murine gammaherpesvirus 68 in vivo system to demonstrate that the E3 ligase Cul4b is essential for this virus to establish latency in germinal center B cells. These findings highlight an essential role for this E3 ligase in promoting chronic gammaherpesvirus infection in vivo and suggest that targeted inhibition of E3 ligases may provide a novel and effective intervention strategy against gammaherpesvirus-associated diseases.

Gammaherpesvirus-mediated repression reveals EWSR1 to be a negative regulator of B cell responses.

The germinal center (GC) plays a central role in the generation of antigen-specific B cells and antibodies. Tight regulation of the GC is essential due to the inherent risks of tumorigenesis and autoimmunity posed by inappropriate GC B cell processes. Gammaherpesviruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) utilize numerous armaments to drive infected naïve B cells, independent of antigen, through GC reactions to expand the latently infected B cell population and establish a stable latency reservoir. We previously demonstrated that the MHV68 microRNA (miRNA) mghv-miR-M1-7-5p represses host EWSR1 (Ewing sarcoma breakpoint region 1) to promote B cell infection. EWSR1 is a transcription and splicing regulator that is recognized for its involvement as a fusion protein in Ewing sarcoma. A function for EWSR1 in B cell responses has not been previously reported. Here, we demonstrate that 1) B cell-specific deletion of EWSR1 had no effect on generation of mature B cell subsets or basal immunoglobulin levels in naïve mice, 2) repression or ablation of EWSR1 in B cells promoted expansion of MHV68 latently infected GC B cells, and 3) B cell-specific deletion of EWSR1 during a normal immune response to nonviral antigen resulted in significantly elevated numbers of antigen-specific GC B cells, plasma cells, and circulating antibodies. Notably, EWSR1 deficiency did not affect the proliferation or survival of GC B cells but instead resulted in the generation of increased numbers of precursor GC B cells. Cumulatively, these findings demonstrate that EWSR1 is a negative regulator of B cell responses.

Transcriptome network data in larval zebrafish (Danio rerio) following exposure to the phenylpyrazole fipronil.

Fipronil is a phenylpyrazole pesticide that is used in both residential and agricultural applications. Fipronil is detected in run-off and water systems that are near areas in which the pesticide has been applied. The pesticide acts to antagonize gamma aminobutyric acid receptors, leading to over-excitation in the central nervous system. Fipronil has relatively high toxicity to fish, but the mechanisms underlying the toxicity are not well understood in embryonic stages. Zebrafish embryos were exposed to a single concentration of fipronil for 48 h at ∼3-4 h-post-fertilization. Following a 7-day depuration phase, transcriptome and behavioral analyses were conducted. Transcriptomics identified neural processes as those differentially expressed with different doses of fipronil (0.2 µg, 200 µg and 2 mg fipronil/L). Gene networks associated with astrocyte differentiation, myelination, neural tube development, brain stem response, innervation, nerve regeneration, astrocyte differentiation, among other pathways were altered with exposure. In addition, miRNA-related events are disrupted by fipronil exposure and genes associated with primary or pri-miRNA processing were increased in larval fish exposed to the pesticide. These data present putative mechanisms associated with neurological impacts at later ages of zebrafish. This is important because it is not clear how early exposure to pesticides like fipronil affect central nervous system function and organisms later in life.

Residual molecular and behavioral effects of the phenylpyrazole pesticide fipronil in larval zebrafish (Danio rerio) following a pulse embryonic exposure.

Pesticides are typically applied to crops as acute applications, and residual effects of such intermittent exposures are not often characterized in developing fish. Fipronil is an agricultural pesticide that inhibits γ-amino-butyric acid (GABA) gated chloride channels. In this study, zebrafish (Danio rerio) embryos were exposed for 48 h (starting at ~3 h post fertilization, hpf) to various concentrations of fipronil (0.02 µg/L up to 4000 µg/L). Following this acute exposure, a subset of fish was transferred to clean water for a 7-day depuration phase. We hypothesized that a pulse exposure to fipronil during critical periods of central nervous system development would adversely affect fish later in life. After a 48 hour pulse exposure, survival was reduced in embryos exposed to 2 µg fipronil/L or greater. However, there was no further mortality during the depuration phase, nor were there changes in body length nor notochord length in larvae 9 dpf (days post-fertilization) compared to controls. Additional experiments were carried out at higher concentrations over 96 h (up to 4 dpf) to also elucidate developmental effects and teratogenicity of fipronil (43.7 µg/L up to 4370 µg/L). Fipronil at these higher concentrations significantly impacted the development of zebrafish, and the following morphometric and teratogenic effects were observed in 4 dpf fish; reduced body length, yolk sac and pericardial edema, reduced midbrain length, reduced optic and otic diameter, and truncation of the lower jaw. In depurated fish, we hypothesized that there would exist residual effects of exposure at the molecular level. Transcriptome profiling was therefore conducted on 9 dpf depurated larvae exposed initially for 48 h to one dose of either 0.2 µg/L, 200 µg/L or 2000 µg/L fipronil. The expression of gene networks associated with glycogen and omega-3-fatty acid metabolism were decreased in larvae exposed to each of the three concentrations of fipronil, suggesting metabolic disruption. Moreover, transcriptomics revealed that fipronil suppressed gene networks related to light-dark adaptation, photoperiod sensing, and circadian rhythm. Based on these data, we tested fish for altered behavioral responses in a Light-Dark preference test. Larvae exposed to >200 µg fipronil/L as embryos showed fewer number of visits (20-30% less) to the dark zone compared to controls. Larvae also spent a lower amount of time in the dark zone compared to controls, suggesting that fipronil strengthened dark avoidance behavior which is indicative of anxiety. This study demonstrates that a short pulse exposure to fipronil can affect transcriptome networks for metabolism, circadian rhythm, and response to light in fish after depuration, and these molecular responses are hypothesized to be related to aberrant behavioral effects observed in the light-dark preference test.

Twenty years of transcriptomics, 17alpha-ethinylestradiol, and fish.

In aquatic toxicology, perhaps no pharmaceutical has been investigated more intensely than 17alpha-ethinylestradiol (EE2), the active ingredient of the birth control pill. At the turn of the century, the fields of comparative endocrinology and endocrine disruption research witnessed the emergence of omics technologies, which were rapidly adapted to characterize potential hazards associated with exposures to environmental estrogens, such as EE2. Since then, significant advances have been made by the scientific community, and as a result, much has been learned about estrogen receptor signaling in fish from environmental xenoestrogens. Vitellogenin, the egg yolk precursor protein, was identified as a major estrogen-responsive gene, establishing itself as the premier biomarker for estrogenic exposures. Omics studies have identified a plethora of estrogen responsive genes, contributing to a wealth of knowledge on estrogen-mediated regulatory networks in teleosts. There have been ~40 studies that report on transcriptome responses to EE2 in a variety of fish species (e.g., zebrafish, fathead minnows, rainbow trout, pipefish, mummichog, stickleback, cod, and others). Data on the liver and testis transcriptomes dominate in the literature and have been the subject of many EE2 studies, yet there remain knowledge gaps for other tissues, such as the spleen, kidney, and pituitary. Inter-laboratory genomics studies have revealed transcriptional networks altered by EE2 treatment in the liver; networks related to amino acid activation and protein folding are increased by EE2 while those related to xenobiotic metabolism, immune system, circulation, and triglyceride storage are suppressed. EE2-responsive networks in other tissues are not as comprehensively defined which is a knowledge gap as regulated networks are expected to be tissue-specific. On the horizon, omics studies for estrogen-mediated effects in fish include: (1) Establishing conceptual frameworks for incorporating estrogen-responsive networks into environmental monitoring programs; (2) Leveraging in vitro and computational toxicology approaches to identify chemicals associated with estrogen receptor-mediated effects in fish (e.g., male vitellogenin production); (3) Discovering new tissue-specific estrogen receptor signaling pathways in fish; and (4) Developing quantitative adverse outcome pathway predictive models for estrogen signaling. As we look ahead, research into EE2 over the past several decades can serve as a template for the array of hormones and endocrine active substances yet to be fully characterized or discovered.

Identification of murine gammaherpesvirus 68 miRNA-mRNA hybrids reveals miRNA target conservation among gammaherpesviruses including host translation and protein modification machinery.

Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish lifelong latent infection in B cells and are associated with a variety of tumors. In addition to protein coding genes, these viruses encode numerous microRNAs (miRNAs) within their genomes. While putative host targets of EBV and KSHV miRNAs have been previously identified, the specific functions of these miRNAs during in vivo infection are largely unknown. Murine gammaherpesvirus 68 (MHV68) is a natural pathogen of rodents that is genetically related to both EBV and KSHV, and thus serves as an excellent model for the study of EBV and KSHV genetic elements such as miRNAs in the context of infection and disease. However, the specific targets of MHV68 miRNAs remain completely unknown. Using a technique known as qCLASH (quick crosslinking, ligation, and sequencing of hybrids), we have now identified thousands of Ago-associated, direct miRNA-mRNA interactions during lytic infection, latent infection and reactivation from latency. Validating this approach, detailed molecular analyses of specific interactions demonstrated repression of numerous host mRNA targets of MHV68 miRNAs, including Arid1a, Ctsl, Ifitm3 and Phc3. Notably, of the 1,505 MHV68 miRNA-host mRNA targets identified in B cells, 86% were shared with either EBV or KSHV, and 64% were shared among all three viruses, demonstrating significant conservation of gammaherpesvirus miRNA targeting. Pathway analysis of MHV68 miRNA targets further revealed enrichment of cellular pathways involved in protein synthesis and protein modification, including eIF2 Signaling, mTOR signaling and protein ubiquitination, pathways also enriched for targets of EBV and KSHV miRNAs. These findings provide substantial new information about specific targets of MHV68 miRNAs and shed important light on likely conserved functions of gammaherpesvirus miRNAs.

Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68.

The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome.

Gammaherpesvirus Readthrough Transcription Generates a Long Non-Coding RNA That Is Regulated by Antisense miRNAs and Correlates with Enhanced Lytic Replication In Vivo.

Gammaherpesviruses, including the human pathogens Epstein⁻Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long non-coding RNAs (lncRNAs). Likewise, the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many as yet uncharacterized lncRNAs. Murine gammaperherpesvirus 68 (MHV68, MuHV-4, HV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. Through the integrated use of parallel data sets from multiple sequencing platforms, we previously resolved transcripts throughout the MHV68 genome, including at least 144 novel transcript isoforms. Here, we sought to molecularly validate novel transcripts identified within the M3/M2 locus, which harbors genes that code for the chemokine binding protein M3, the latency B cell signaling protein M2, and 10 microRNAs (miRNAs). Using strand-specific northern blots, we validated the presence of M3-04, a 3.91 kb polyadenylated transcript that initiates at the M3 transcription start site and reads through the M3 open reading frame (ORF), the M3 poly(a) signal sequence, and the M2 ORF. This unexpected transcript was solely localized to the nucleus, strongly suggesting that it is not translated and instead may function as a lncRNA. Use of an MHV68 mutant lacking two M3-04-antisense pre-miRNA stem loops resulted in highly increased expression of M3-04 and increased virus replication in the lungs of infected mice, demonstrating a key role for these RNAs in regulation of lytic infection. Together these findings suggest the possibility of a tripartite regulatory relationship between the lncRNA M3-04, antisense miRNAs, and the latency gene M2.

Reprint of: Environmental toxicology and omics: A question of sex.

Molecular initiating events and downstream transcriptional/proteomic responses provide valuable information for adverse outcome pathways, which can be used predict the effects of chemicals on physiological systems. There has been a paucity of research that addresses sex-specific expression profiling in toxicology and due to cost, time, and logistic considerations, sex as a variable has not been widely considered. In response to this deficiency, federal agencies in the United States, Canada, and Europe have highlighted the importance of including sex as a variable in scientific investigations. Using case studies from both aquatic and mammalian toxicology, we report that there can be less than ~20-25% consensus in how the transcriptome and proteome of each sex responds to chemicals. Chemicals that have been shown to elicit sex-specific responses in the transcriptome or proteome include pharmaceuticals, anti-fouling agents, anticorrosive agents, and fungicides, among others. Sex-specific responses in the transcriptome and proteome are not isolated to whole animals, as investigations demonstrate that primary cell cultures isolated from each sex responds differently to toxicants. This signifies that sex is important, even in cell lines. Sex has significant implications for predictive toxicology, and both male and female data are required to improve robustness of adverse outcome pathways. BIOLOGICAL SIGNIFICANCE: Clinical toxicology recognizes that sex is an important variable, as pharmacokinetics (ADME; absorption, distribution, metabolism, and excretion) can differ between females and males. However, few studies in toxicology have explored the implication of sex in relation to the transcriptome and proteome of whole organisms. High-throughput molecular approaches are becoming more frequently applied in toxicity screens (e.g. pre-clinical experiments, fish embryos, cell lines, synthetic tissues) and such data are expected to build upon reporter-based cell assays (e.g. receptor activation, enzyme inhibition) used in toxicant screening programs (i.e. Tox21, ToxCast, REACH). Thus, computational models can more accurately predict the diversity of adverse effects that can occur from chemical exposure within the biological system. Our studies and those synthesized from the literature suggest that the transcriptome and proteome of females and males respond quite differentially to chemicals. This has significant implications for predicting adverse effects in one sex when using molecular data generated in the other sex. While molecular initiating events are not expected to differ dramatically between females and males (i.e. an estrogen binds estrogen receptors in both sexes), it is important to acknowledge that the downstream transcriptomic and proteomic responses can differ based upon the presence/absence of co-regulators and inherent sex-specific variability in regulation of transcriptional and translational machinery. Transcriptomic and proteomic studies also reveal that cell processes affected by chemicals can differ due to sex, and this can undoubtedly lead to sex-specific physiological responses.

Environmental toxicology and omics: A question of sex.

Molecular initiating events and downstream transcriptional/proteomic responses provide valuable information for adverse outcome pathways, which can be used predict the effects of chemicals on physiological systems. There has been a paucity of research that addresses sex-specific expression profiling in toxicology and due to cost, time, and logistical considerations, sex as a variable has not been widely considered. In response to this deficiency, federal agencies in the United States, Canada, and Europe have highlighted the importance of including sex as a variable in scientific investigations. Using case studies from both aquatic and mammalian toxicology, we report that there can be less than ~20-25% consensus in how the transcriptome and proteome of each sex responds to chemicals. Chemicals that have been shown to elicit sex-specific responses in the transcriptome or proteome include pharmaceuticals, anti-fouling agents, anticorrosive agents, and fungicides, among others. Sex-specific responses in the transcriptome and proteome are not isolated to whole animals, as investigations demonstrate that primary cell cultures isolated from each sex responds differently to toxicants. This signifies that sex is important, even in cell lines. Sex has significant implications for predictive toxicology, and both male and female data are required to improve robustness of adverse outcome pathways. BIOLOGICAL SIGNIFICANCE: Clinical toxicology recognizes that sex is an important variable, as pharmacokinetics (ADME; absorption, distribution, metabolism, and excretion) can differ between females and males. However, few studies in toxicology have explored the implication of sex in relation to the transcriptome and proteome of whole organisms. High-throughput molecular approaches are becoming more frequently applied in toxicity screens (e.g. pre-clinical experiments, fish embryos, cell lines, synthetic tissues) and such data are expected to build upon reporter-based cell assays (e.g. receptor activation, enzyme inhibition) used in toxicant screening programs (i.e. Tox21, ToxCast, REACH). Thus, computational models can more accurately predict the diversity of adverse effects that can occur from chemical exposure within the biological system. Our studies and those synthesized from the literature suggest that the transcriptome and proteome of females and males respond quite differently to chemicals. This has significant implications for predicting adverse effects in one sex when using molecular data generated in the other sex. While molecular initiating events are not expected to differ dramatically between females and males (i.e. an estrogen binds estrogen receptors in both sexes), it is important to acknowledge that the downstream transcriptomic and proteomic responses can differ based upon the presence/absence of co-regulators and inherent sex-specific variability in regulation of transcriptional and translational machinery. Transcriptomic and proteomic studies also reveal that cell processes affected by chemicals can differ due to sex, and this can undoubtedly lead to sex-specific physiological responses.

Estrogen-responsive gene networks in the teleost liver: What are the key molecular indicators?

An overarching goal of environmental genomics is to leverage sensitive suites of markers that are robust and reliable to assess biological responses in a range of species inhabiting variable environments. The objective of this study was to identify core groups of transcripts and molecular signaling pathways that respond to 17alpha-ethylinestadiol (EE2), a ubiquitous estrogenic contaminant, using transcriptome datasets generated from six independent laboratories. We sought to determine which biomarkers and gene networks were those most robust and reliably detected in multiple laboratories. Six laboratories conducted microarray analysis in pieces of the same liver from male fathead minnows exposed to ∼15ng/L EE2 for 96h. There were common transcriptional networks identified in every dataset. These included down-regulation of gene networks associated with blood clotting, complement activation, triglyceride storage, and xenobiotic metabolism. Noteworthy was that more than ∼85% of the gene networks were suppressed by EE2. Leveraging both these data and those mined from the Comparative Toxicogenomics Database (CTD), we narrowed in on an EE2-responsive transcriptional network. All transcripts in this network responded ∼±5-fold or more to EE2, increasing reliability of detection. This network included estrogen receptor alpha, transferrin, myeloid cell leukemia 1, insulin like growth factor 1, insulin like growth factor binding protein 2, and methionine adenosyltransferase 2A. This estrogen-responsive interactome has the advantage over single markers (e.g. vitellogenin) in that these entities are directly connected to each other based upon evidence of expression regulation and protein binding. Thus, it represents an interacting functional suite of estrogenic markers. Vitellogenin, the gold standard for estrogenic exposures, can show high individual variability in its response to estrogens, and the use of a multi-gene approach for estrogenic chemicals is expected to improve sensitivity. In our case, the coefficient of variation was significantly lowered by the gene network (∼67%) compared to Vtg alone, supporting the use of this transcriptional network as a sensitive alternative for detecting estrogenic effluents and chemicals. We propose that screening chemicals for estrogenicity using interacting genes within a defined expression network will improve sensitivity, accuracy, and reduce the number of animals required for endocrine disruption assessments.

How consistent are we? Interlaboratory comparison study in fathead minnows using the model estrogen 17α-ethinylestradiol to develop recommendations for environmental transcriptomics.

Fundamental questions remain about the application of omics in environmental risk assessments, such as the consistency of data across laboratories. The objective of the present study was to determine the congruence of transcript data across 6 independent laboratories. Male fathead minnows were exposed to a measured concentration of 15.8 ng/L 17α-ethinylestradiol (EE2) for 96 h. Livers were divided equally and sent to the participating laboratories for transcriptomic analysis using the same fathead minnow microarray. Each laboratory was free to apply bioinformatics pipelines of its choice. There were 12 491 transcripts that were identified by one or more of the laboratories as responsive to EE2. Of these, 587 transcripts (4.7%) were detected by all laboratories. Mean overlap for differentially expressed genes among laboratories was approximately 50%, which improved to approximately 59.0% using a standardized analysis pipeline. The dynamic range of fold change estimates was variable between laboratories, but ranking transcripts by their relative fold difference resulted in a positive relationship for comparisons between any 2 laboratories (mean R2 > 0.9, p < 0.001). Ten estrogen-responsive genes encompassing a fold change range from dramatic (>20-fold; e.g., vitellogenin) to subtle (∼2-fold; i.e., block of proliferation 1) were identified as differentially expressed, suggesting that laboratories can consistently identify transcripts that are known a priori to be perturbed by a chemical stressor. Thus, attention should turn toward identifying core transcriptional networks using focused arrays for specific chemicals. In addition, agreed-on bioinformatics pipelines and the ranking of genes based on fold change (as opposed to p value) should be considered in environmental risk assessment. These recommendations are expected to improve comparisons across laboratories and advance the use of omics in regulations. Environ Toxicol Chem 2017;36:2593-2601. © 2017 SETAC.

Molecular initiating events of the intersex phenotype: Low-dose exposure to 17α-ethinylestradiol rapidly regulates molecular networks associated with gonad differentiation in the adult fathead minnow testis.

Intersex, or the presence of oocytes in the testes, has been documented in fish following exposure to wastewater effluent and estrogenic compounds. However, the molecular networks underlying the intersex condition are not completely known. To address this, we exposed male fathead minnows to a low, environmentally-relevant concentration of 17alpha-ethinylestradiol (EE2) (15ng/L) and measured the transcriptome response in the testis after 96h to identify early molecular initiating events that may proceed the intersex condition. The short-term exposure to EE2 did not affect gonadosomatic index and proportion of gametes within the testes. However, the production of 11-ketotestosterone and testosterone from the testis in vitro was decreased relative to controls. Expression profiling using a 8×60K fathead minnow microarray identified 10 transcripts that were differentially expressed in the testes, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase). Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

Transcriptional networks associated with the immune system are disrupted by organochlorine pesticides in largemouth bass (Micropterus salmoides) ovary.

Largemouth bass (Micropterus salmoides) inhabiting Lake Apopka, Florida are exposed to high levels of persistent organochlorine pesticides (OCPs) and dietary uptake is a significant route of exposure for these apex predators. The objectives of this study were to determine the dietary effects of two organochlorine pesticides (p, p'-dichlorodiphenyldichloroethylene; p, p' DDE and methoxychlor; MXC) on the reproductive axis of largemouth bass. Reproductive bass (late vitellogenesis) were fed one of the following diets: control pellets, 125ppm p, p'-DDE, or 10ppm MXC (mg/kg) for 84days. Due to the fact that both p,p' DDE and MXC have anti-androgenic properties, the anti-androgenic pharmaceutical flutamide was fed to a fourth group of largemouth bass (750ppm). Following a 3 month exposure, fish incorporated p,p' DDE and MXC into both muscle and ovary tissue, with the ovary incorporating 3 times more organochlorine pesticides compared to muscle. Endpoints assessed were those related to reproduction due to previous studies demonstrating that these pesticides impact the reproductive axis and we hypothesized that a dietary exposure would result in impaired reproduction. However, oocyte distribution, gonadosomatic index, plasma vitellogenin, and plasma sex steroids (17ß-estradiol, E2 and testosterone, T) were not different between control animals and contaminant-fed largemouth bass. Moreover, neither p, p' DDE nor MXC affected E2 or T production in ex vivo oocyte cultures from chemical-fed largemouth bass. However, both pesticides did interfere with the normal upregulation of androgen receptor that is observed in response to human chorionic gonadotropin in ex vivo cultures, an observation that may be related to their anti-androgenic properties. Transcriptomics profiling in the ovary revealed that gene networks related to cell processes such as leukocyte cell adhesion, ossification, platelet function and inhibition, xenobiotic metabolism, fibrinolysis, and thermoregulation were altered by p, p' DDE, MXC, and flutamide. Interestingly, immune-related gene networks were suppressed by all three chemicals. The data suggest that p, p' DDE and flutamide affected more genes in common with each other than either chemical with MXC, consistent with studies suggesting that p, p' DDE is a more potent anti-androgen than MXC. These data demonstrate that reproductive health was not affected by these specific dietary treatments, but rather the immune system, which may be a significant target of organochlorine pesticides. The interaction between the reproductive and immune systems should be considered in future studies on these legacy and persistent pesticides.

Defining the role of omics in assessing ecosystem health: Perspectives from the Canadian environmental monitoring program.

Scientific reviews and studies continue to describe omics technologies as the next generation of tools for environmental monitoring, while cautioning that there are limitations and obstacles to overcome. However, omics has not yet transitioned into national environmental monitoring programs designed to assess ecosystem health. Using the example of the Canadian Environmental Effects Monitoring (EEM) program, the authors describe the steps that would be required for omics technologies to be included in such an established program. These steps include baseline collection of omics endpoints across different species and sites to generate a range of what is biologically normal within a particular ecosystem. Natural individual variability in the omes is not adequately characterized and is often not measured in the field, but is a key component to an environmental monitoring program, to determine the critical effect size or action threshold for management. Omics endpoints must develop a level of standardization, consistency, and rigor that will allow interpretation of the relevance of changes across broader scales. To date, population-level consequences of routinely measured endpoints such as reduced gonad size or intersex in fish is not entirely clear, and the significance of genome-wide molecular, proteome, or metabolic changes on organism or population health is further removed from the levels of ecological change traditionally managed. The present review is not intended to dismiss the idea that omics will play a future role in large-scale environmental monitoring studies, but rather outlines the necessary actions for its inclusion in regulatory monitoring programs focused on assessing ecosystem health.

Transcript variability and physiological correlates in the fathead minnow ovary: Implications for sample size, and experimental power.

Fundamental studies characterizing transcript variability in teleost tissues are needed if molecular endpoints are to be useful for regulatory ecotoxicology. The objectives of this study were to (1) measure transcript variability of steroidogenic enzymes and steroid receptors in the fathead minnow (FHM; Pimephales promelas) ovary to better determine normal variability and the sample sizes needed to detect specific effect sizes and to (2) determine how expression patterns related to higher level endpoints used in some regulatory ecotoxicology programs (e.g. relative gonad size). Estrogen receptor 2b (esr2b) and 5α-reductase a3 (srd5a3) showed high variability in the ovary (CV>1.0) while progesterone receptor (pgr), androgen receptor (ar), and esr2a showed comparatively low variability (CV=~0.5--0.7). Using these estimates, a power analysis revealed that sample sizes for real-time PCR experiments would need to be>20 to detect a 2-fold change for 7 of the transcripts examined; thus many molecular studies conducted in the fish ovary may have insufficient power to detect smaller effects. Two transcripts were correlated to steroid production in the ovary; cyp19a1 levels were positively correlated to in vitro E2 production, while ar levels were negatively correlated to in vitro T production. Thus, these transcripts may be informative molecular surrogates for ovarian steroid production. No transcript investigated showed any correlation to GSI, condition, or body weight/length. Molecular approaches in fish are increasingly used to assess biological impacts of chemical stressors; however additional studies are required that determine how molecular variability relates to higher level biological endpoints.

Progesterone increases ex vivo testosterone production and decreases the expression of progestin receptors and steroidogenic enzymes in the fathead minnow (Pimephales promelas) ovary.

Progesterone (P4) is a metabolic precursor for a number of steroids, including estrogens and androgens. P4 also has diverse roles within the vertebrate ovary that include oocyte growth and development. The objectives of this study were to measure the effects of P4 on testosterone (T) and 17ß-estradiol (E2) production in the fathead minnow (FHM) ovary and on the mRNA abundance of transcripts involved in steroidogenesis and steroid receptor signaling. Ovary explants were treated with P4 (10(-6)M) for 6 and 12h. P4 administration significantly increased T production ∼3-fold at both 6 and 12h, whereas E2 production was not affected, consistent with the hypothesis that excess P4 is not converted to terminal estrogens in the mature ovary. Nuclear progesterone receptor mRNA was decreased at 6h and membrane progesterone receptor gamma-2 mRNA was significantly down-regulated at both 6 and 12h; however there was no change in membrane progesterone receptor alpha or beta mRNA levels. Androgen receptor (ar) and estrogen receptor 2a (esr2a) mRNA were significantly reduced at 6h with P4 treatment, but there was no change in esr2b mRNA at either time point. Transcripts for enzymes in the steroid pathway (star, hsd11b2) were significantly lower at 6h compared to controls, whereas cyp17a and cyp19a mRNA abundance did not change with treatments at either time point. These data suggest that P4 incubation can lead to increased T production in the FHM ovary without a concomitant change in E2, and that the membrane bound progestin receptors are differentially regulated by P4 in the teleost ovary. As environmental progestins have received increased attention due to their suspected role as endocrine disruptors, mechanistic data on the role of exogenous P4 treatments in the male and female gonad is warranted.

An inter-laboratory study on the variability in measured concentrations of 17ß-estradiol, testosterone, and 11-ketotestosterone in white sucker: implications and recommendations.

Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish, potentially by altering circulating concentrations of 17ß-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT). Common methods to measure steroids in plasma samples include radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs). The present study examines variability in E2, T, and 11-KT across 8 laboratories measuring reference and pulp mill effluent-exposed white sucker (Catostomus commersoni) plasma. We examine the contribution of assay type (RIA vs ELISA), standardized hormone extraction, location of values on the standard curve (upper and lower limits), and other variables on the ability to distinguish hormone levels between reference and exposed fish and the impact of these variables on quantitation of hormones in different laboratories. Of the 8 participating laboratories, 7 of 8 and 7 of 7 identified differences between sites for female E2 and female T, respectively, and 7 of 7 and 4 of 5 identified no differences between male T and male 11-KT. Notably, however, the ng/mL concentration of steroids measured across laboratories varied by factors of 10-, 6-, 14-, and 10-fold, respectively. Within laboratory intra-assay variability was generally acceptable and below 15%. Factors contributing to interlaboratory variability included calculation errors, assay type, and methodology. Based on the interlaboratory variability detected, we provide guidelines and recommendations to improve the accuracy and precision of steroid measurements in fish ecotoxicology studies.

Transcriptomic profiling of progesterone in the male fathead minnow (Pimephales promelas) testis.

P4 is a hormone with diverse functions that include roles in reproduction, growth, and development. The objectives of this study were to examine the effects of P4 on androgen production in the mature teleost testis and to identify molecular signaling cascades regulated by P4 to improve understanding of its role in male reproduction. Fathead minnow (FHM) testis explants were treated in vitro with two concentrations of P4 (10(-8) and 10(-6) M) for 6 and 12 h. P4 significantly increased testosterone (T) production in the FHM testis but did not affect 11-ketotestosterone. Gene network analysis revealed that insulin growth factor (Igf1) and tumor necrosis factor receptor (Tnfr) signaling was significantly depressed with P4 treatment after 12h. There was also a 20% increase in a gene network for follicle-stimulating hormone secretion and an 18% decrease in genes involved in vasopressin signaling. Genes in steroid metabolism (e.g. star, cyp19a, 11bhsd) were not significantly affected by P4 treatments in this study, and it is hypothesized that pre-existing molecular machinery may be more involved in the increased production of T rather than the de novo expression of steroid-related transcripts and receptors. There was a significant decrease in prostaglandin E synthase 3b (cytosolic) (ptges3b) after treatment with P4, suggesting that there is cross talk between P4 and prostaglandin pathways in the reproductive testis. P4 has a role in regulating steroid production in the male testis and may do so by modulating gene networks related to endocrine pathways, such as Igf1, Tnfr, and vasopressin.

Protein targets of acrylamide adduct formation in cultured rat dopaminergic cells.

Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate groups on cysteine residues of neuronal proteins. Identifying specific proteins targeted by ACR can lead to a better mechanistic understanding of the corresponding neurotoxicity. Therefore, in the present study, the ACR-adducted proteome in exposed primary immortalized mesencephalic dopaminergic cells (N27) was determined using tandem mass spectrometry (LTQ-Orbitrap). N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics identified a total of 15,243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys groups. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity, diabetes, inflammation, nerve degeneration and atherosclerosis. These results provide detailed information regarding the ACR-adducted proteome in a dopaminergic cell line. The catalog of affected proteins indicates the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the corresponding neurotoxic mechanism.