Nancy Denslow

In this paper existing regulatory frameworks and test systems for assessing potential endocrine-active chemicals are described, and associated challenges discussed, along with proposed approaches to address these challenges. Regulatory frameworks vary somewhat across geographies, but all basically evaluate whether a chemical possesses endocrine activity and whether this activity can result in adverse outcomes either to humans or the environment. Current test systems include in silico, in vitro and in vivo techniques focused on detecting potential endocrine activity, and in vivo tests that collect apical data to detect possible adverse effects. These test systems are currently designed to robustly assess endocrine activity and/or adverse effects in the estrogen, androgen, and thyroid hormone signaling pathways; however, there are some limitations of current test systems for evaluating endocrine hazard and risk. These limitations include a lack of certainty regarding: 1) adequately se...

Transcriptomic analysis can complement traditional ecotoxicology data by providing mechanistic insight, and by identifying sub-lethal organismal responses and contaminant classes underlying observed toxicity. Before transcriptomic information can be used in monitoring and risk assessment, it is necessary to determine its reproducibility and detect key steps impacting the reliable identification of differentially expressed genes. A custom 15K-probe microarray was used to conduct transcriptomics analyses across six laboratories with estuarine amphipods exposed to cyfluthrin-spiked or control sediments (10 days). Two sample types were generated, one consisted of total RNA extracts (Ex) from exposed and control samples (extracted by one laboratory) and the other consisted of exposed and control whole body amphipods (WB) from which each laboratory extracted RNA. Our findings indicate that gene expression microarray results are repeatable. Differentially expressed data had a higher degree of repeatability across all laboratories in samples with similar RNA quality (Ex) when compared to WB samples with more variable RNA quality. Despite such variability a subset of genes were consistently identified as differentially expressed across all laboratories and sample types. We found that the differences among the individual laboratory results can be attributed to several factors including RNA quality and technical expertise, but the overall results can be improved by following consistent protocols and with appropriate training.

Smallmouth bass (Micropterus dolomieu) were collected to quantify the nature and prevalence of biomarker responses, including biochemical indices, toxicopathic lesions and general health indices, among fish collected from polychlorinated biphenyl (PCB)-contaminated and nearby uncontaminated reaches of the Kalamazoo River, Michigan, USA. Blood and tissue samples (gill, liver, spleen, head kidney, trunk kidney, thyroid and gonads) were collected and preserved at necropsy for biochemical and histological analyses. The body condition factor and liver somatic index were significantly lower in fish collected from the downstream, contaminated site. Plasma vitellogenin was not detected in male fish collected from either site. Liver ethoxyresorufin-O-deethylase activity and liver and spleen superoxide dismutase activity were significantly depressed in fish collected from the downstream site. Significant toxicopathic lesions such as glycogen depletion, enhanced macrophage aggregates, hepatic foci of cellular alteration (i.e. preneoplastic lesions) and neoplasia were also detected in the liver of fish collected from the downstream site. This study indicates that many of the biochemical and histopathological biomarker responses were associated with liver and body tissue PCB concentrations. Taken together, the biomarkers of exposure and effect strongly suggest that fish within the downstream site are adversely affected by PCBs and other chemical stressors.

The pathogenesis of inflammatory periodontal disease was studied by examining the mechanism of HeLa and HL60 cell growth inhibition by cell-free saline-soluble extracts of Eikenella corrodens and bacterial plaque. Previous studies identified a protein (p80) as causing growth inhibition by E. corrodens extracts. After purification by two-dimensional SDS-PAGE, p80 was digested with protease lysC. Amino acid sequences were obtained and

... Ozols, J. (1990) Meth Enzymol. 182, (Deutscher, MP ed.) Academic Press, San Deigo pp. 587-601. ... Gharahdgaghi, F., Atherton, D., DeMott, M., and Mische SM (1992) in "Techniques in Protein Chemistry III" (RH Angeletti, ed.) Academic Press, San Diego, pp 249-260 Excerpt. ...

The mission of protected areas is to conserve biodiversity and improve human welfare. To assess the effect of urban waters entering into protected areas, we performed 48-h whole-effluent exposures with fathead minnows, analyzing changes in steady state levels of mRNAs in the livers of exposed fish. Raw wastewater, treated city wastewater, and treated wastewater from a university were collected for exposures. All exposed fish showed altered mRNA levels of DNA damage-repair genes. Fish exposed to raw and treated wastewaters showed down-regulation of transcripts for key intermediates of cholesterol biosynthesis and elevated plasma cholesterol. The type of wastewater treatment influenced the response of gene transcription. Because of the relevance of some of the altered cellular pathways, we suggest that these effluents may cause deleterious effects on fish inside protected areas that receive these waters. Inclusion of research and mitigation efforts for this type of threat in protected areas management is advised.

The estrogenicity of a municipal wastewater effluent was monitored using the vitellogenin biomarker in adult male fathead minnows (Pimephales promelas). The variability in the expression of vitellogenin was evident among the monitoring periods. Significant (α⩽0.05) increases in plasma vitellogenin concentrations were detected in March and December, but not in August or June. Additionally, the magnitude of expression was variable. Variability

A variety of anthropogenic chemicals are capable of binding to the estrogen receptor of vertebrate species. Binding of these compounds can interfere with homeostasis by disrupting normal gene expression patterns. The purpose of this study was to investigate the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in coastal habitats

A variety of anthropogenic compounds are capable of binding to the estrogen receptor (ER) of vertebrate species. Binding of these chemicals to the ER can interfere with homeostasis by altering normal gene expression patterns. The purpose of this study was to characterize the expression of 30 genes using a sheepshead minnow (Cyprinodon variegatus) cDNA macroarray. Many of the genes on the array were previously identified by differential display reverse transcriptase-polymerase chain reaction to be upregulated or downregulated in sheepshead minnows treated through aqueous exposure to known or suspected estrogenic chemicals. The results of this study show that 17 beta-estradiol (E2), 17 alpha-ethinyl estradiol (EE2), diethylstilbestrol (DES), and methoxychlor (MXC) have similar genetic signatures for the 30 genes examined. The genetic signature of fish treated with p-nonylphenol was identical in pattern to that in fish treated with E2, EE2, DES, and MXC except for the additional upregulation of a cDNA clone that shares similarity to ubiquitin-conjugating enzyme 9. Endosulfan produced results that resembled the gene expression patterns of untreated control fish with exception of the upregulation of estrogen receptor alpha and the downregulation of a cDNA clone that shares similarity to 3-hydroxy-3-methylglutaryl-coenzyme A reductase. We show that our estrogen-responsive cDNA macroarray can detect dose-dependent changes in gene expression patterns in fish treated with EE2.

Male summer flounder (Paralichthys dentatus) were given two injections (initially and 2 weeks later) of 17β-estradiol (E2) totaling 0.2 (2×0.1), 2.0 (2×1.0) or 20.0 (2×10.0) mg E2/kg body weight. Blood and tissue samples were collected 4, 6 and 8 weeks after the initial injection in the (2×0.1) mg/kg treatment, 4, 6, 8, and 15 weeks after the first injection in

During the last decade there has been a significant body of research conducted on environmental estrogens. These include industrial, agricultural and pest-control chemicals that bind to the estrogen receptor and induce biological changes during development or reproduction. Most of these changes are probably due to modified gene expression, since estrogen receptors function at this level. We have mapped qualitative gene

Five natural, pharmaceutical, or xenobiotic chemicals [17β-estradiol (E2), ethynylestradiol (EE2), diethystilbestrol (DES), methoxychlor (MXC), nonylphenol (NP)] were tested in two in vitro assays [yeast estrogen screen (YES), MCF-7 breast tumor cell proliferation (E-Screen)], and compared with previously reported results from two in vivo male sheepshead minnow vitellogenin (VTG) production studies. The purpose of this investigation was to determine how accurately

Fish were collected from 16 sites on rivers in the Columbia River Basin (CRB) from September 1997 to April 1998 to document temporal and spatial trends in the concentrations of accumulative contaminants and to assess contaminant effects on the fish. Sites were located on the mainstem of the Columbia River and on the Snake, Willamette, Yakima, Salmon, and Flathead Rivers. Common carp (Cyprinus carpio), black bass (Micropterus sp.), and largescale sucker (Catostomus macrocheilus) were the targeted species. Fish were field-examined for external and internal lesions, selected organs were weighed to compute somatic indices, and tissue and fluid samples were preserved for fish health and reproductive biomarker analyses. Composite samples of whole fish, grouped by species and gender, from each site were analyzed for organochlorine and elemental contaminants using instrumental methods and for 2,3,7,8-tetrachloro dibenzo-p-dioxin-like activity (TCDD-EQ) using the H4IIE rat hepatoma cell bioassay. Overall, pesticide concentrations were greatest in fish from lower CRB sites and elemental concentrations were greatest in fish from upper CRB sites. These patterns reflected land uses. Lead (Pb) concentrations in fish from the Columbia River at Northport and Grand Coulee, Washington (WA) exceeded fish and wildlife toxicity thresholds (>0.4 microg/g). Selenium (Se) concentrations in fish from the Salmon River at Riggins, Idaho (ID), the Columbia River at Vernita Bridge, WA, and the Yakima River at Granger, WA exceeded toxicity thresholds for piscivorous wildlife (>0.6 microg/g). Mercury (Hg) concentrations in fish were elevated throughout the basin but were greatest (>0.4 microg/g) in predatory fish from the Salmon River at Riggins, ID, the Yakima River at Granger, WA, and the Columbia River at Warrendale, Oregon (OR). Residues of p,p'-DDE were greatest (>0.8 microg/g) in fish from agricultural areas of the Snake, Yakima, and Columbia River basins but were not detected in upper CRB fish. Other organochlorine pesticides did not exceed toxicity thresholds in fish or were detected infrequently. Total polychlorinated biphenyls (PCBs; >0.11 microg/g) and TCDD-EQs (>5 pg/g) exceeded wildlife guidelines in fish from the middle and lower CRB, and ethoxyresorufin O-deethylase (EROD) activity was also elevated at many of the same sites. Temporal trend analysis indicated decreasing or stable concentrations of Pb, Se, Hg, p,p'-DDE, and PCBs at most sites where historical data were available. Altered biomarkers were noted in fish throughout the CRB. Fish from some stations had responded to chronic contaminant exposure as indicated by fish health and reproductive biomarker results. Although most fish from some sites had grossly visible external or internal lesions, histopathological analysis determined these to be inflammatory responses associated with helminth or myxosporidian parasites. Many largescale sucker from the Columbia River at Northport and Grand Coulee, WA had external lesions and enlarged spleens, which were likely associated with infections. Intersex male smallmouth bass (Micropterus dolomieu) were found in the Snake River at Lewiston, ID and the Columbia River at Warrendale, OR. Male bass, carp, and largescale sucker containing low concentrations of vitellogenin were common in the CRB, and comparatively high concentrations (>0.3 mg/mL) were…

A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta-elimination and Michael addition chemistries in a 'one-step' reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution- or gel-based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low-abundance post-labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation-site mapping has been evaluated for a phosphoprotein standard, bovine beta-casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT-labeled proteins or peptides.

Queen conch (Strombus gigas) reproduction is inhibited in nearshore areas of the Florida Keys, relative to the offshore environment where conchs reproduce successfully. Nearshore reproductive failure is possibly a result of exposure to environmental factors, including heavy metals, which are likely to accumulate close to shore. Metals such as Cu and Zn are detrimental to reproduction in many mollusks. Histology shows gonadal atrophy in nearshore conchs as compared to reproductively healthy offshore conchs. In order to determine molecular mechanisms leading to tissue changes and reproductive failure, a microarray was developed. A normalized cDNA library for queen conch was constructed and sequenced using the 454 Life Sciences GS-FLX pyrosequencer, producing 27,723 assembled contigs and 7,740 annotated transcript sequences. The resulting sequences were used to design the microarray. Microarray analysis of conch testis indicated differential regulation of 255 genes (p<0.01) in nearshore conch, relative to offshore. Changes in expression for three of four transcripts of interest were confirmed using real-time reverse transcription polymerase chain reaction. Gene Ontology enrichment analysis indicated changes in biological processes: respiratory chain (GO:0015992), spermatogenesis (GO:0007283), small GTPase-mediated signal transduction (GO:0007264), and others. Inductively coupled plasma-mass spectrometry analysis indicated that Zn and possibly Cu were elevated in some nearshore conch tissues. Congruence between testis histology and microarray data suggests that nearshore conch testes regress during the reproductive season, while offshore conch testes develop normally. Possible mechanisms underlying the testis regression observed in queen conch in the nearshore Florida Keys include a disruption of small GTPase (Ras)-mediated signaling in testis development. Additionally, elevated tissue levels of Cu (34.77 ng/mg in testis) and Zn (831.85 ng/mg in digestive gland, 83.96 ng/mg in testis) nearshore are similar to reported levels resulting in reproductive inhibition in other gastropods, indicating that these metals possibly contribute to NS conch reproductive failure.

... DOI: 10.1080/02755947.2011.591231 Jo Ellen Hinck a , Diana M. Papoulias a , Mandy L. Annis a ... Biomarker differences among reproductive categories were tested with analysis of variance (ANOVA) using Fisher's unrestricted least significant difference (Saville 199044. ...

Androgenic chemicals are present in the environment at concentrations that impair reproductive processes in fish. The objective of this experiment was to identify proteins and cell processes mediated through androgen receptor signaling using an androgen receptor agonist (17beta-trenbolone) and antagonist (flutamide) in the liver. Female fathead minnows were exposed to nominal concentrations of either 17beta-trenbolone (0.05, 0.5, or 5 microg/L), flutamide (50, 150, or 500 microg/L), or a mixture (500 microg flutamide/L and 0.5 microg 17beta-trenbolone/L) for 48 h. The iTRAQ method was used to label peptides after protein extraction and trypsin-digestion from livers of untreated controls or from fish treated with 17beta-trenbolone (5 microg/L), flutamide (500 microg/L), or a mixture of both compounds. Forty-five proteins were differentially altered by one or more treatments (p<0.05). Many altered proteins were involved in cellular metabolism (e.g., glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase), general and oxidative stress response (e.g., superoxide dismutase and heat shock proteins), and the regulation of translation (e.g., ribosomal proteins). Cellular pathway analysis identified additional signaling cascades activated or inhibited by flutamide that may not be androgen receptor mediated. We also compared changes in select proteins to changes in their mRNA levels and observed, in general, that proteins and mRNA changes did not correlate, suggesting complex regulation at the level of both the transcriptome and proteome. It is concluded that both transcriptomic and proteomic approaches offer unique and complementary insights into mechanisms of regulation. We demonstrate the utility of proteomic profiling for use on a model species with value to ecotoxicology but having limited genomic information.