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Nancy D Denslow

Publications and source records attributed to Nancy D Denslow.

At least 19 recordsLinked to original sources

Exposure to p,p'-DDE or dieldrin during the reproductive season alters hepatic CYP expression in largemouth bass (Micropterus salmoides).

Largemouth bass (LMB) in Central Florida living on sites with high levels of organochlorine pesticides (OCPs) have exhibited poor reproductive success and altered steroid profiles. The mechanism underlying these changes is unknown, however changes in the rate of steroid metabolism could alter steroid homeostasis. Members of the CYP2 and CYP3A families play a significant role in the metabolism of many xenobiotics and endogenous compounds, including sex steroids. Therefore, the goal of this study was to identify members of the CYP2 and CYP3A families in LMB and characterize the effects of OCP exposure on their expression. Full-length clones of two CYP3A isoforms were obtained from LMB liver, CYP3A68 and 3A69, which exhibited significant sequence divergence. Full-length clones for CYP2N14 and CYP2P11 were also obtained from LMB liver. Steady-state mRNA levels of each of these CYPs increased in both sexes between early reproductive phase (December) and peak reproductive phase (March). Expression of CYP3A68 and CYP2P11 was sexually dimorphic during peak reproductive phase with 2-fold higher expression in females and males, respectively. Foodborne exposure to 46 ppm p,p'-DDE or 0.8 ppm dieldrin for 30 days did not have a significant effect on expression of CYPs. However, 4 months exposure to p,p'-DDE induced CYP3A68 and 3A69 expression in both sexes, while dieldrin produced weak induction of CYP3A68 and suppressed CYP3A69 expression in females, but had no effect on males. Neither p,p'-DDE nor dieldrin significantly altered the expression of CYP2P11 or CYP2N14. This work demonstrates that there are significant changes in CYP expression that occur during LMB reproduction which can be modified by exposure to OCPs.

Amino Acid Sequence↗

Temporal gene induction patterns in sheepshead minnows exposed to 17beta-estradiol.

Gene arrays provide a powerful method to examine changes in gene expression in fish due to chemical exposures in the environment. In this study, we expanded an existing gene array for sheepshead minnows (Cyprinodon variegatus) (SHM) and used it to examine temporal changes in gene expression for male SHM exposed to 100 ng 17beta-estradiol (E(2))/L for five time points between 0 and 48 hr. We found that in addition to the induction of genes involved in oocyte development (vitellogenin [VTG], zona radiata [ZRP]), other genes involved in metabolism and the inflammatory response are also affected. We identified five patterns of temporal induction in genes whose expression was modified due to E(2) exposure. We validated the gene array data for the expression of VTG 1, VTG 2, ZRP 2 and ZRP 3 and found that with low levels of exogenous E(2) (100 ng E(2)/L) exposure, ZRP expression precedes VTG expression. However, at higher concentrations of E(2) (500 ng E(2)/L), the difference in temporal expression appears to be lost. Exposure to high levels of environmental contaminants may affect the normal ordered expression of genes required for reproduction. Gene expression profiling using arrays promises to be a valuable tool in the field of environmental toxicology. As more genes are identified for species used in toxicological testing, researchers will be better able to predict adverse effects to chemical exposures and to understand the relationships between changes in gene expression and changes in phenotype.

Animals↗

Novel differential neuroproteomics analysis of traumatic brain injury in rats.

Approximately two million traumatic brain injury (TBI) incidents occur annually in the United States, yet there are no specific therapeutic treatments. The absence of brain injury diagnostic endpoints was identified as a significant roadblock to TBI therapeutic development. To this end, our laboratory has studied mechanisms of cellular injury for biomarker discovery and possible therapeutic strategies. In this study, pooled naïve and injured cortical samples (48 h postinjury; rat controlled cortical impact model) were processed and analyzed using a differential neuroproteomics platform. Protein separation was performed using combined cation/anion exchange chromatography-PAGE. Differential proteins were then trypsinized and analyzed with reversed-phase LC-MSMS for protein identification and quantitative confirmation. The results included 59 differential protein components of which 21 decreased and 38 increased in abundance after TBI. Proteins with decreased abundance included collapsin response mediator protein 2 (CRMP-2), glyceraldehyde-3-phosphate dehydrogenase, microtubule-associated proteins MAP2A/2B, and hexokinase. Conversely C-reactive protein, transferrin, and breakdown products of CRMP-2, synaptotagmin, and alphaII-spectrin were found to be elevated after TBI. Differential changes in the above mentioned proteins were confirmed by quantitative immunoblotting. Results from this work provide insight into mechanisms of traumatic brain injury and yield putative biochemical markers to potentially facilitate patient management by monitoring the severity, progression, and treatment of injury.

Animals↗

Dietary exposure of largemouth bass to OCPs changes expression of genes important for reproduction.

Dieldrin and p,p'-DDE are ubiquitous contaminants known to act as endocrine disruptors, causing impaired development and reproduction in fish and wildlife. In order to elucidate the mechanisms by which dieldrin and p,p'-DDE cause endocrine disruption in largemouth bass (Micropterus salmoides), fish were exposed subchronically through the diet to both contaminants. Following 120 days of exposure, p,p'-DDE decreased estradiol in females, but increased 11-ketotestosterone in both sexes. Dieldrin on the other hand, decreased estradiol and 11-ketotestosterone in both sexes. Both pesticides also altered steady state mRNA expression levels of a set of genes chosen to represent three possible mechanisms of endocrine disruption: (1) direct interaction with soluble sex steroid receptors, (2) biosynthesis of endogenous sex hormones, and (3) metabolism of endogenous hormones. p,p'-DDE acted as a weak estrogen, increasing the expression of vitellogenin and estrogen receptor alpha in the liver. p,p'-DDE also altered the expression of genes involved in the synthesis of endogenous hormones as well as their metabolism. Dieldrin, on the other hand, only altered expression of vitellogenin and not estrogen receptor alpha. Dieldrin also altered the expression of genes involved in hormone synthesis and metabolism, and it dramatically lowered plasma hormone levels. Both pesticides targeted expression of genes involved in all three modes of action, suggesting that they each have multiple modes of action.

Animals↗

Environmental contaminants and biomarker responses in fish from the Columbia River and its tributaries: spatial and temporal trends.

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 measured in male fish from the Flathead River at Creston, Montana, the Snake River at Ice Harbor Dam, WA, and the Columbia River at Vernita Bridge, WA and Warrendale, OR. Results from our study and other investigations indicate that continued monitoring in the CRB is warranted to identify consistently degraded sites and those with emerging problems.

Animals↗

A multidimensional differential proteomic platform using dual-phase ion-exchange chromatography-polyacrylamide gel electrophoresis/reversed-phase liquid chromatography tandem mass spectrometry.

Differential proteomic analysis has arisen as a large-scale means to discern proteome-wide changes upon treatment, injury, or disease. Tandem protein separation methods are required for large-scale differential proteomic analysis. Here, a novel multidimensional platform for resolving and differentially analyzing complex biological samples is presented. The platform, collectively termed CAX-PAGE/RPLC-MSMS, combines biphasic ion-exchange chromatography with polyacrylamide gel electrophoresis for protein separation, quantification, and differential band targeting, followed by capillary reversed-phase liquid chromatography and data-dependent tandem mass spectrometry for quantitative and qualitative peptide analysis. CAX-PAGE provides high protein resolving power with a theoretical peak capacity of 3570, extendable to 7600, a wide protein mass range verified from 16 to 273 kDa, and reproducible differential sample comparison without the added expense of fluorescent dyes and imaging equipment. Demonstrated using a neuroproteomic model, CAX-PAGE revealed an increased number of differential proteins, 137, compared with 82 found by 2D difference gel electrophoresis. When combined with RPLC-MSMS for protein identification, an additional quantification step is performed for internal validation, confirming a 2-fold or greater change in 89% of identified differential targets.

Animals↗

Environmental contaminants and biomarker responses in fish from the Rio Grande and its U.S. tributaries: spatial and temporal trends.

We collected, examined, and analyzed 368 fish of seven species from 10 sites on rivers of the Rio Grande Basin (RGB) during late 1997 and early 1998 to document temporal and geographic trends in the concentrations of accumulative contaminants and to assess contaminant effects on the fish. Sites were located on the mainstem of the Rio Grande and on the Arroyo Colorado and Pecos River in Texas (TX), New Mexico (NM), and Colorado. Common carp (Cyprinus carpio) and largemouth bass (Micropterus salmoides) were the targeted species. Fish were examined in the field for internal and external visible gross lesions, selected organs were weighed to compute ponderal and organosomatic indices, and samples of tissues and fluids were obtained and preserved for analysis of fish health and reproductive biomarkers. Whole fish from each station were composited by species and gender and analyzed for organochlorine chemical residues 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, fish from lower RGB stations contained greater concentrations of organochlorine pesticide residues and appeared to be less healthy than those from sites in the central and upper parts of the basin, as indicated by a general gradient of residue concentrations and biomarker responses. A minimal number of altered biomarkers and few or no elevated contaminant concentrations were noted in fish from the upper RGB. The exception was elevated concentrations [up to 0.46 microg/g wet-weight (ww)] of total mercury (Hg) in predatory species from the Rio Grande at Elephant Butte Reservoir, NM, a condition documented in previous studies. Arsenic (As) and selenium (Se) concentrations were greatest in fish from sites in the central RGB; Se concentrations in fish from the Pecos River at Red Bluff Lake, TX and from the Rio Grande at Langtry, TX and Amistad International Reservoir, TX exceeded published fish and wildlife toxicity thresholds. In the lower RGB, residues of p,p'-DDT metabolites (<or=1.69 microg/g ww), chlordane-related compounds (<or=0.21 microg/g ww), dieldrin (<or=0.0.05 microg/g ww), and toxaphene (<or=2.4 microg/g ww) were detected in fish from most sites; maximum concentrations were in channel catfish (Ictalurus punctatus) from the Arroyo Colorado at Harlingen, TX. Concentrations of one or more residues exceeded toxicity thresholds for fish and wildlife in fish from this site and from the Rio Grande at Mission, TX and Brownsville, TX; however, concentrations were lower than those reported by previous studies. In addition, the proportional concentrations of p,p'-DDT at all sites were low, indicating weathered DDT rather than the influx of new material. Concentrations of total PCBs (<0.05 microg/g ww) and TCDD-EQ (<or=6 pg/g ww) were comparatively low in all samples. Hepatic ethoxyresorufin O-deethylase (EROD) activity in some fish was elevated relative to reference rates at most sites, but was generally lower than previously reported activity in fish from heavily contaminated locations. The comparatively low PCB and TCDD-EQ concentrations together with elevated EROD activity may reflect exposure to polycyclic aromatic hydrocarbons. Reproductive biomarkers were consistent with chronic contaminant exposure at lower RGB sites; comparatively large percentages of intersex male largemouth bass, relatively low gonadosomatic indices, and elevated plasma vitellogenin concentrations in male fish were noted at three of the four stations. Large percentages of atretic eggs were also observed in the ovaries of female common carp from the Rio Grande at Brownsville, TX. Although many of the conditions noted may have other causes in addition to contaminant exposure, the biomarker results for the lower RGB sites are consistent with subtle responses of fish to contaminants, an interpretation supported by the chemical data of this and other investigations.

Animals↗

Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry.

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.

Affinity Labels↗

Rapid discovery of putative protein biomarkers of traumatic brain injury by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry.

We report the rapid discovery of putative protein biomarkers of traumatic brain injury (TBI) by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry (SDS-PAGE-Capillary LC-MS(2)). Ipsilateral hippocampus (IH) samples were collected from naive rats and rats subjected to controlled cortical impact (a rodent model of TBI). Protein database searching with 15,558 uninterpreted MS(2) spectra, collected in 3 days via data-dependent capillary LC-MS(2) of pooled cyanine dye-labeled samples separated by SDS-PAGE, identified more than 306 unique proteins. Differential proteomic analysis revealed differences in protein sequence coverage for 170 mammalian proteins (57 in naive only, 74 in injured only, and 39 of 64 in both), suggesting these are putative biomarkers of TBI. Confidence in our results was obtained by the presence of several known biomarkers of TBI (including alphaII-spectrin, brain creatine kinase, and neuron-specific enolase) in our data set. These results show that SDS-PAGE prior to in vitro proteolysis and capillary LC-MS(2) is a promising strategy for the rapid discovery of putative protein biomarkers associated with a specific physiological state (i.e., TBI) without a priori knowledge of the molecules involved.

Amino Acid Sequence↗

Gene expression fingerprints of largemouth bass (Micropterus salmoides) exposed to pulp and paper mill effluents.

Effluents from pulp and paper mills that historically have used elemental chlorine in the bleaching process have been implicated in inhibiting reproduction in fish. Compounds with estrogenic and androgenic binding affinities have been found in these effluents, suggesting that the impairment of reproduction is through an endocrine-related mode of action. To date, a great deal of attention has been paid to phytoestrogens and resin acids that are present in mill process streams as a result of pulping trees. Estrogen and estrogen mimics interact directly with the estrogen receptor and have near immediate effects on gene transcription by turning on the expression of a unique set of genes. Using differential display (DD) RT-PCR, we examined changes in gene expression induced by exposure to paper mill effluents. Largemouth bass were exposed to 0, 10, 20, 40, and 80% paper mill effluent concentrations in large flow-through tanks for varied periods of time including 7, 28 or 56 days. Plasma hormone levels in males and females and plasma vitellogenin (Vtg) in females decreased with dose and time. Measurements of changes in gene expression using DD RT-PCR suggest that the gene expression patterns of male fish do not change much with exposure, except for the induction of a few genes including CYP 1A, a protein that is induced through the action of the Ah receptor in response to dioxin and similar polyaromatic hydrocarbons. However, in the case of females, exposure to these effluents resulted in an up-regulation of CYP 1A that was accompanied by a generalized down-regulation of genes normally expressed during the reproductive season. These antiestrogenic changes are in agreement with previous studies in bass exposed to these effluents, and could result in decreased reproductive success in affected populations.

Amino Acid Sequence↗

Differential expression of largemouth bass (Micropterus salmoides) estrogen receptor isotypes alpha, beta, and gamma by estradiol.

The expression levels of three estrogen receptor (ER) isotypes alpha, beta, and gamma were quantified in female largemouth bass (Micropterus salmoides) (LMB) liver, ovary, brain, and pituitary tissues. ER alpha and beta expression predominated in the liver, while ERs beta and gamma predominated in the other tissues. Temporally in females, ER alpha was highly up-regulated, ER gamma was slightly up-regulated, and ER beta levels remained unchanged in the liver when plasma 17-beta estradiol (E2) and vitellogenin (Vtg) levels were elevated in the spring. In ovarian tissue from these same fish, all three ERs were maximally expressed in the fall, during early oocyte development and prior to peak plasma E2 levels. When males were injected with E2, ER alpha was highly inducible, ER gamma was moderately up-regulated, and ER beta levels were not affected. None of the ER isotypes were induced by E2 in gonadal tissues. These results combined suggest that the ERs themselves are not regulated in the same manner by E2, and furthermore, do not contribute equally to the transcriptional regulation of genes involved in fish reproduction such as Vtg.

Amino Acid Sequence↗

Relationship between reproductive success and male plasma vitellogenin concentrations in cunner, Tautogolabrus adspersus.

The gene for vitellogenin, an egg yolk protein precursor, is usually silent in male fish but can be induced by estrogen exposure. For this reason, vitellogenin production in male fish has become a widely used indicator of exposure to exogenous estrogens or estrogen mimics in the aquatic environment. The utility of this indicator to predict impacts on fish reproductive success is unclear because information on the relationship between male plasma vitellogenin and reproductive end points in male and female fish is limited. In the research reported in this article, we investigated whether the presence of male plasma vitellogenin is a reliable indicator of decreased reproductive success in mature fish. Adult and sexually mature male and female cunner (Tautogolabrus adspersus) were exposed to 17ss-estradiol, ethynylestradiol, or estrone, three steroidal estrogens that elicit the vitellogenic response. Data were gathered and pooled on egg production, egg viability, egg fertility, sperm motility, and male plasma vitellogenin concentrations. All males, including two with plasma vitellogenin levels exceeding 300 mg/mL, produced motile sperm. Neither percent fertile eggs nor percent viable eggs produced by reproductively active fish demonstrated a significant correlation with male plasma vitellogenin concentrations. Male gonadosomatic index and average daily egg production by females showed significant, but weak, negative correlation with male plasma vitellogenin concentrations. Results suggest that male plasma vitellogenin expression is not a reliable indicator of male reproductive dysfunction in adult cunner exposed to estrogens for 2-8 weeks during their reproductive season, at least in relation to capacity to produce motile sperm or fertilize eggs. Male plasma vitellogenin expression may serve as an indicator of reduced female reproductive function caused by estrogen exposure.

Animals↗

Effects of pulp and paper mill effluents on reproductive success of largemouth bass.

This study evaluated the effects of bleached and unbleached kraft mill effluent on reproductive success of largemouth bass (Micropterus salmoides). Bass were exposed to effluent concentrations (0, 10, 20, 40, or 80%) for 28 and 56 d. Parameters measured included hepatosomatic index (HSI) and gonadosomatic index (GSI) and plasma concentrations of 17beta-estradiol (E2), 11-ketotestosterone (11-KT), and vitellogenin (VTG). At the end of the 56-d period, bass were moved to hatchery ponds to evaluate spawning success. Spawning mats with eggs either were brought indoors for evaluation of fecundities, hatchabilities, and egg and fry size (measured at age 3 d), or were left in ponds and fry number and size recorded (average age of 14 d). Effluent exposure was verified by measuring resin acids (isopimaric, abietic. and dehydroabietic acids) in bile. Compared to controls, exposed bass had greater concentrations of resin acids in bile. In general, exposed females had lower concentrations of E2 and VTG (> or = 20% effluent), whereas males had lower concentrations of 11-KT (> or = 20% effluent) and increased E2 (> or = 20% effluent). The HSI values increased in females (> or = 10% effluent), and GSI values decreased in both sexes (> or = 40% effluent). Fecundity, egg size, and hatchability did not differ across treatments, but an increase in the frequency of fry abnormalities and a decrease in fry weights was observed at effluent exposures of 40% and higher. However, results from the pond study, revealed a significant reduction in fry growth and survival (> or = 10%). This decline may have been caused by an increased frequency of deformities, in conjunction with alterations of growth. These changes could have resulted from alterations in egg quality because of failure of parental reproductive systems, from acute embryo toxicity after translocation of contaminants from the mother to the developing embryo, or from both.

Animals↗

Expression profiling of estrogenic compounds using a sheepshead minnow cDNA macroarray.

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.

Animals↗

Estradiol-induced gene expression in largemouth bass (Micropterus salmoides).

Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E(2)). Single injections of E(2) ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER alpha sequences. After acute E(2)-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E(2) in largemouth bass and that for Vtg a delayed primary response. The specific effect of E(2) on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen gamma was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E(2) involves a complex network of responses over time.

Amino Acid Sequence↗

A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro.

Five natural, pharmaceutical, or xenobiotic chemicals [17beta-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 the two in vitro assays predicted responses observed in live animals. EC50 values for all five chemicals were approximately one order of magnitude less sensitive in the YES assay than in the MCF-7 assay. Based on the EC50 values, DES was 1.1 (YES) to 2.5 (MCF-7) times more potent in these receptor binding assays than was E2, while EE2 was slightly less potent than E2 in the YES assay (0.7) and nearly twice as potent (1.9) as E2 in the MCF-7 assay. EE2 and DES were of approximately equal potency in the 13-day sheepshead minnow VTG production bioassay. Both MXC and NP were 10(7) times less potent than E2 in the YES assay, MXC was 10(5) times less estrogenic than E2 in the MCF-7 assay, while both were approximately 100 times less potent than E2 in the live animal bioassay. The in vitro tests were substantially less sensitive (at least 1000 times) than the sheepshead minnow VTG assay for estimating estrogenic potency of the two xenobiotic chemicals, which suggests that in vitro-based, large-scale screening programs could potentially result in many false negative evaluations.

Animals↗

An evaluation of biomarkers of reproductive function and potential contaminant effects in Florida largemouth bass ( Micropterus salmoidesfloridanus) sampled from the St. Johns River.

The objective of this study was to describe and compare several reproductive parameters for Florida largemouth bass (Micropterus salmoides floridanus) inhabiting the St. Johns River and exposed to different types and/or degrees of contamination. Welaka was selected as the reference site in this study because of its low urban and agricultural development, Palatka is in close proximity to a paper mill plant, the Green Cove site is influenced by marine shipping activities and Julington Creek site receives discharges of domestic wastewater and storm water runoff from recreational boating marinas. For this study, bass were sampled both prior to (September 1996) and during the spawning season (February 1997). In order to characterize chemical exposure, bass livers were analyzed for up to 90 trace organics and 11 trace metal contaminants. Reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads and plasma concentrations of vitellogenin (VTG), 17beta-estradiol (E2) and 11-ketotestosterone (11-KT). In general, the sum of organic chemicals was highest in livers from Palatka bass and bass from Green Cove and Julington Creek had higher hepatic concentrations of low molecular polycyclic aromatic hydrocarbons and polychlorinated biphenyls when compared to fish from Welaka. Metals were more variable across sites, with highest mean concentrations found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or Welaka (Cd, Hg, Pb, Se, Tn). Female bass from Palatka and Green Cove had lower concentrations of E2, VTG and lower GSI in relation to Welaka. Males from Palatka and Green Cove showed comparable declines in 11-KT in relation to males from Julington Creek and GSI were decreased only in Palatka males. These results indicate a geographical trend in reproductive effects, with changes being most pronounced at the site closest to the paper mill (Palatka) and decreasing as the St. Johns River flows downstream. Since reproductive alterations were most evident in bass sampled from the site closest to the paper mill discharge, it is possible that exposure to these effluents might explain at least some of the results reported here. However, the presence of reproductive alterations in fish sampled at a considerable distance from the mill discharge (Green Cove, 40 km) would suggest exposure to chemicals released from sources other than the paper mill plant. It is clear that additional studies are needed to evaluate the potential impact of these reproductive changes in populations of Florida largemouth bass inhabiting the St. Johns River.

Animals↗