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The Canadair Defence Systems Division (DSD) is responsible for making all major repairs and modifications on the Canadian fighter CF-18. Although the work varies, DSD must establish a maintenance program each year that specifies estimated starting and ending dates for every maintenance project. It modifies this program constantly during the year because of discrepancies between planned and actual project duration. To improve the accuracy of its plan, DSD used simulation to assess the influence of uncertainty on project delivery dates. Managers can now rely on an accurate risk-analysis tool when making strategic decisions.

Lincoln Creek is a severely degraded urban stream located in Milwaukee County, Wisconsin, USA. As part of a comprehensive study on effects of urban storm water runoff on the stream biota, an in vitro bioassay with PLHC-1 (Poeciliopsis lucida) fish hepatoma cells was used to assess potential toxic potency of aryl hydrocarbon receptor (AhR)-active compounds, collected by semipermeable membrane devices (SPMDs) exposed to Lincoln Creek water. Dialysates from SPMDs exposed to Lincoln Creek water caused marked cytochrome P4501A induction in PLHC-1. Toxic potency of dialysates, expressed as bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) ranged from 1,300 to 6,600 pg TCDD-EQ/g SPMD for 14-d exposures. Dialysates from SPMDs exposed to stream water at base flow had potencies consistently lower than those exposed to storm-flow (high-flow) events that occurred during the same 14-d period. Polychlorinated biphenyls were not detectable in the dialysates. Gas chromatography-mass spectrometry analysis identified polycyclic aromatic hydrocarbons (PAHs) as major contaminants in the dialysates. A log-log correlation of total PAHs and TCDD-EQ yielded an r2 of 0.802. Empirical evidence suggests that AhR-active PAHs can account for about 20 to 50% of the potency observed.

Spironolactone is a pharmaceutical that in humans is used to treat conditions like hirsutism, various dermatologic afflictions, and female-pattern hair loss through antagonism of the androgen receptor. Although not routinely monitored in the environment, spironolactone has been detected downstream of a pharmaceutical manufacturer, indicating a potential for exposure of aquatic species. Furthermore, spironolactone has been reported to cause masculinization of female western mosquitofish, a response indicative of androgen receptor activation. Predictive methods to identify homologous proteins to the human and western mosquitofish androgen receptor suggest that vertebrates would be more susceptible to adverse effects mediated by chemicals like spironolactone that target the androgen receptor compared with invertebrate species that lack a relevant homolog. In addition, an adverse outcome pathway previously developed for activation of the androgen receptor suggests that androgen mimics can lead to reproductive toxicity in fish. To assess this, 21-d reproduction studies were conducted with 2 fish species, fathead minnow and Japanese medaka, and the invertebrate Daphnia magna. Spironolactone significantly reduced the fecundity of medaka and fathead minnows at 50 μg/L, whereas daphnia reproduction was not affected by concentrations as large as 500 μg/L. Phenotypic masculinization of females of both fish species was observed at 5 μg/L as evidenced by formation of tubercles in fathead minnows and papillary processes in Japanese medaka. Effects in fish occurred at concentrations below those reported in the environment. These results demonstrate how a priori knowledge of an adverse outcome pathway and the conservation of a key molecular target across vertebrates can be utilized to identify potential chemicals of concern in terms of monitoring and highlight potentially sensitive species and endpoints for testing.

Adaptive or compensatory responses to chemical exposure can significantly influence in vivo concentration-duration-response relationships. This study provided data to support development of a computational dynamic model of the hypothalamic-pituitary-gonadal axis of a model vertebrate and its response to aromatase inhibitors as a class of endocrine active chemicals. Fathead minnows (Pimephales promelas) were either exposed to the aromatase inhibitor fadrozole (0.5 or 30 μg/l) continuously for 1, 8, 12, 16, 20, 24, or 28 days or exposed for 8 days and then held in control water (no fadrozole) for an additional 4, 8, 12, 16, or 20 days. The time course of effects on ovarian steroid production, circulating 17β-estradiol (E2) and vitellogenin (VTG) concentrations, and expression of steroidogenesis-related genes in the ovary was measured. Exposure to 30 μg fadrozole/l significantly reduced plasma E2 and VTG concentrations after just 1 day and those effects persisted throughout 28 days of exposure. In contrast, ex vivo E2 production was similar to that of controls on day 8-28 of exposure, whereas transcripts coding for aromatase and follicle-stimulating hormone receptor were elevated, suggesting a compensatory response. Following cessation of fadrozole exposure, ex vivo E2 and plasma E2 concentrations exceeded and then recovered to control levels, but plasma VTG concentrations did not, even after 20 days of depuration. Collectively these data provide several new insights into the nature and time course of adaptive responses to an aromatase inhibitor that support development of a computational model (see companion article).

Spironolactone is a pharmaceutical that in humans is used to treat conditions like hirsutism, various dermatologic afflictions, and female-pattern hair loss through antagonism of the androgen receptor. Although not routinely monitored in the environment, spironolactone has been detected downstream of a pharmaceutical manufacturer, indicating a potential for exposure of aquatic species. Furthermore, spironolactone has been reported to cause masculinization of female western mosquitofish, a response indicative of androgen receptor activation. Predictive methods to identify homologous proteins to the human and western mosquitofish androgen receptor suggest that vertebrates would be more susceptible to adverse effects mediated by chemicals like spironolactone that target the androgen receptor compared with invertebrate species that lack a relevant homolog. In addition, an adverse outcome pathway previously developed for activation of the androgen receptor suggests that androgen mimics can lead to reproductive toxicity in fish. To assess this, 21-d reproduction studies were conducted with 2 fish species, fathead minnow and Japanese medaka, and the invertebrate Daphnia magna. Spironolactone significantly reduced the fecundity of medaka and fathead minnows at 50 μg/L, whereas daphnia reproduction was not affected by concentrations as large as 500 μg/L. Phenotypic masculinization of females of both fish species was observed at 5 μg/L as evidenced by formation of tubercles in fathead minnows and papillary processes in Japanese medaka. Effects in fish occurred at concentrations below those reported in the environment. These results demonstrate how a priori knowledge of an adverse outcome pathway and the conservation of a key molecular target across vertebrates can be utilized to identify potential chemicals of concern in terms of monitoring and highlight potentially sensitive species and endpoints for testing.

Gamma-aminobutyric acid (GABA) and GABA receptors play an important role in neuroendocrine regulation in fish. Disruption of the GABAergic system by environmental contaminants could interfere with normal regulation of the hypothalamic-pituitary-gonadal axis, leading to impaired fish reproduction. The present study used a 21-d fathead minnow (Pimephales promelas) reproduction assay to investigate the reproductive toxicity of fipronil (FIP), a broad-spectrum phenylpyrazole insecticide that acts as a noncompetitive blocker of GABA receptor-gated chloride channels. Continuous exposure up to 5 µg FIP/L had no significant effect on most of the endpoints measured, including fecundity, secondary sexual characteristics, plasma steroid and vitellogenin concentrations, ex vivo steroid production, and targeted gene expression in gonads or brain. The gonad mass, gonadosomatic index, and histological stage of the gonad were all significantly different in females exposed to 0.5 µg FIP/L compared with those exposed to 5.0 µg FIP/L; however, there were no other significant effects on these measurements in the controls or any of the other treatments in either males and females. Overall, the results do not support a hypothesized adverse outcome pathway linking FIP antagonism of the GABA receptor(s) to reproductive impairment in fish.

The aim of this study was to determine the presence of oxyiminocephalosporin-resistant (OCR) Gram-negative bacilli and extended-spectrum β-lactamase (ESBL)-producing isolates in stool specimens obtained from paediatric patients hospitalised for acute diarrhoea. We conducted a prospective, multicentre study over a period of 6 months in seven hospitals in the south of France. Samplings were carried out from infants admitted for acute diarrhoea with no previous antibiotic treatment in the last week. Bacteria in stool specimens were screened for the presence of OCR Gram-negative bacilli on Drigalski agar supplemented with ceftazidime and ESBL CHROMagar® media, and confirmed by the Rosco tablets test. Genetic detection was performed by the Check MDR® microarray and by polymerase chain reaction (PCR) and sequencing with bacterial DNA extracted from isolates. The presence of OCR enterobacteria was markedly high (177/1,118 patients, 15.2 %), with an important community origin (66.1 %). The majority of multidrug-resistant (MDR) bacteria were Enterobacter cloacae (106, 59.9 %) and Escherichia coli (61, 34.5 %). The prevalence of ESBL and CTX-M producers represented 5.2 and 4.3 % of the isolates, respectively. The main proportion of these ESBL carriers was found in children less than 1 year of age (53.4 %). One carbapenemase (IMP-1) was detected. The study revealed the wide dissemination of MDR bacteria in infants attending hospitals in the south of France during a non-outbreak situation, in particular, the spread of cefotaximase and the detection of a carbapenemase. This worrisome situation must reinforce the use of hygiene procedures and appropriate antibiotics to control the emergence and spread of OCR organisms.

Endocrine disruption is considered a highly relevant hazard for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening tests with a focus on interference with estrogen, androgen, and thyroid hormone pathways in fish and amphibians have been developed. However, they use a large number of animals and short-term alternatives to animal tests would be advantageous. Therefore, the status of alternative assays for endocrine disruption in fish and frogs was assessed by a detailed literature analysis. The aim was to (i) determine the strengths and limitations of alternative assays and (ii) present conclusions regarding chemical specificity, sensitivity, and correlation with in vivo data. Data from 1995 to present were collected related to the detection/testing of estrogen-, androgen-, and thyroid-active chemicals in the following test systems: cell lines, primary cells, fish/frog embryos, yeast and cell-free systems. The review shows that the majority of alternative assays measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis. Other mechanisms were rarely analysed. A database was established and used for a quantitative and comparative analysis. For example, a high correlation was observed between cell-free ligand binding and cell-based reporter cell assays, between fish and frog estrogenic data and between fish embryo tests and in vivo reproductive effects. It was concluded that there is a need for a more systematic study of the predictive capacity of alternative tests and ways to reduce inter- and intra-assay variability.

Widespread environmental contamination by bisphenol A (BPA) has created the need to fully define its potential toxic mechanisms of action (MOA) to properly assess human health and ecological risks from exposure. Although long recognized as an estrogen receptor (ER) agonist, some data suggest that BPA may also behave as an androgen receptor (AR) antagonist. However, direct evidence of this activity is deficient. To address this knowledge gap, we employed a metabolomic approach using in vivo exposures of fathead minnows (FHM; Pimephales promelas ) to BPA either alone or in a binary mixture with 17β-trenbolone (TB), a strong AR agonist. Changes in liver metabolite profiles in female FHM in response to these exposures were determined using high resolution (1)H NMR spectroscopy and multivariate and univariate statistics. Using this approach, we observed clear evidence of the ability of BPA to mitigate the impact of TB, consistent with an antiandrogenic MOA. In addition, a transcriptional activation assay with the FHM AR was used to confirm the AR antagonistic activity of BPA in vitro. The results of these in vivo and in vitro analyses provide strong and direct evidence for ascribing an antiandrogenic MOA to BPA in vertebrates.

Chemical structures of several urinary reproductive pheromones in fish have been identified, and their role in the chemical communication of reproductive condition is well characterized. On the contrary, the role of chemical communication in signalling of social/territorial status in fish is poorly understood. Fathead minnows are an example of a fish species whose life history traits appear conducive to evolution of chemical communication systems that confer information about social/territorial status. Male reproduction in this species is dependent upon their ability to acquire and defend a high quality nesting territory, and to attract a female to the nest. We hypothesized that fathead minnow males use visual and urine-derived chemical cues to signal territorial status. To test this hypothesis, effects of territorial acquisition on male-specific secondary sex characteristics (SSCs) and urine volumes were first assessed. Second, frequencies of male urination in varying social contexts were examined. Finally, nuclear magnetic resonance-based metabolomics was used to identify urinary metabolites that were differentially excreted in the urine of territorial versus non-territorial males. The expression of SSCs, sperm, and urine volumes increased with territory acquisition, and either remained unchanged or decreased in non-territorial males. Frequency of male urination increased significantly in the presence of females (but not males), suggesting that females are the main target of the urinary signals. Territorial and non-territorial males had distinct urinary metabolomic profiles. An unforeseen finding was that one could discern future territorial status of males, based on their initial metabolomic profiles. Bile acids and volatile amines were identified as potential chemical signals of social status in the fathead minnow. The finding that trimethylamine (a fishy smelling volatile amine) may be a social cue is particularly interesting, because it is known to bind trace amine-associated receptors, indicating that these receptors may play role in chemical signalling of social status in fish.

Fibrates are a class of pharmaceuticals that indirectly modulate cholesterol biosynthesis through effects on peroxisome proliferator-activated receptors. Gemfibrozil is a fibrate that has been detected in wastewater treatment plant influents, effluents, and drinking water. The objective of the present study was to assess the potential physiological and reproductive impacts of gemfibrozil on fathead minnows (Pimephales promelas). Fish were exposed to gemfibrozil in two different studies. The first was a short-term test with water concentrations of 0, 15, and 600 µg gemfibrozil/L, sampling after 2 or 8 d of exposure. Plasma cholesterol concentrations were significantly reduced in males exposed to 600 µg gemfibrozil/L for 8 d. In addition, expression of several hepatic genes important to lipid metabolism was altered, suggesting that gemfibrozil does affect lipid metabolism in fish. A 21-d study was conducted to investigate further the effects on lipid metabolism and steroidogenesis as well as to assess potential impacts of gemfibrozil on reproduction. Fish were exposed to water concentrations of 0, 1.5, 15, 600, and 1,500 µg gemfibrozil/L. Exposure to 1,500 µg gemfibrozil/L caused a modest, but not significant, reduction in fecundity. However, gemfibrozil had no consistent effect on plasma cholesterol, triglycerides, or sex steroids after 21 d of exposure. The present study showed no evidence for significant physiological or reproductive impacts of gemfibrozil at an environmentally relevant concentration of 1.5 µg/L.

The objective of this study was to evaluate temporal effects of the model steroidogenesis inhibitor ketoconazole (KTC) on aspects of reproductive endocrine function controlled by the hypothalamic-pituitary-gonadal (HPG) axis in the fathead minnow ( Pimephales promelas ). Ketoconazole inhibits the activity of two cytochrome P450s (CYPs) key to sex steroid production in vertebrates, CYP11a (cholesterol side chain cleavage) and CYP17 (c17α-hydroxylase/17, 20-lyase). Sexually mature fish were exposed to water-borne KTC (30 or 300 μg/L) in a flow-through system for up to 8 d, following which animals were allowed to recover in clean water. Fish were sampled after 1, 4 and 8 d of exposure, and after 1, 8 and 16 d of recovery. A shorter-term time-course experiment also was conducted in which females were sampled on seven occasions during a 12 h KTC exposure. Ketoconazole consistently depressed ex vivo gonadal synthesis of testosterone (T) in both sexes, and 17β-estradiol (E2) in females during both exposure and recovery phases of the time-course studies. Effects on ex vivo steroidogenesis in females occurred within as little as 1 h of exposure. Plasma concentrations of T in males and E2 in females also were depressed by exposure to KTC, but these decreases did not persist to the same degree as observed for the ex vivo effects. In females, after decreases within 12 h, plasma E2 concentrations were similar to (or greater than) controls at 24 h of exposure, while in males, plasma T returned to levels comparable to controls within 1 d of cessation of KTC exposure. The discrepancy between the ex vivo and in vivo data at later stages in the test is consistent with some type of compensatory response to KTC in fish. However, we were unable to ascertain the mechanistic basis for such a response. For example, although a number of genes related to steroid synthesis ( e.g. , cyp11a , cyp17 ) were up-regulated in the gonads of both males and females during the exposure and early recovery phases of the experiment, this did not seem to account for the resurgence in plasma steroid concentrations in KTC-exposed fish. Further studies focused on metabolism and clearance of steroids might lend insights as to the effects of KTC on plasma steroid concentrations. Overall, our results demonstrate the complex, temporally dynamic nature of the vertebrate HPG system in response to chemical stressors.

This study examined the ability of environmentally relevant concentrations of 10 different polybrominated diphenyl ethers (PBDEs) to induce aryl hydrocarbon receptor (AhR)- and estrogen receptor (ER)-mediated gene expression in vitro. It also examined the ability of PBDEs to displace steroid hormones from serum proteins in vitro. At concentrations ranging up to 880 ng/ ml, none of the PBDEs significantly displaced tritiated 17beta-estradiol (E2) or testosterone from hormone-stripped carp serum. At concentrations ranging up to 500 ng/ml, 9 of 10 PBDEs tested failed to induce ER- or AhR-mediated gene expression in MVLN and H4IIE-luc cells, respectively. One congener, 3,3',4,4',5-pentabromodiphenylether (BDE 126), induced significant AhR-mediated gene expression at 500 ng/ml, but the magnitude of induction was only 13% of that caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Overall, the PBDEs tested were found to be at least 200,000 times less potent than TCDD and 50,000 times less potent than E2 for inducing AhR- and ER-mediated gene expression, respectively.

The epidemiology of staphylococcal community-acquired skin and soft tissues infections (CA-SSTIs) has changed dramatically. We described prospectively the characteristics of the Staphylococcus aureus isolated from 71 non-teaching French hospitals and implicated in CA-SSTIs: antimicrobial susceptibility (mecA polymerase chain reaction [PCR], disk diffusion method), virulence factor gene (sea, tst, pvl) prevalence and genetic background (agr allele). During November 2006, 235 strains were collected (wound infection: 51%, abscess: 21%, whitlow: 8%, diabetic foot: 7%, furunculosis: 3%). sea, tst and pvl were identified in 22.1, 13.2 and 8.9% strains, respectively. agr allele 1 was the most frequently encountered genetic background, whatever the methicillin susceptibility. Among the 34 methicillin-resistant S. aureus (MRSA, 14.5% of all S. aureus), only one strain (2.9%) harboured pvl (belonging to the European ST80 clone), four (11.8%) tst (belonging to two endemic French clones) and 18 (52.9%) sea gene (mainly the Lyon clone). According to their in vitro activity, pristinamycin or trimethoprim/sulfamethoxazole could be considered as first-choice antibiotics. To date, the international pvl-positive MRSA clones have not spread in France. MRSA strains isolated from putative CA-SSTIs exhibited a genetic and phenotypic background of hospital-acquired (HA) clones. National survey should be continued, in order to monitor the emergence of virulent clones.

Effects of bisphenol A (BPA) on ovarian transcript profiles as well as targeted end points with endocrine/reproductive relevance were examined in two fish species, fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), exposed in parallel using matched experimental designs. Four days of waterborne exposure to 10 μg BPA/L caused significant vitellogenin induction in both species. However, zebrafish were less sensitive to effects on hepatic gene expression and steroid production than fathead minnow and the magnitude of vitellogenin induction was more modest (i.e., 3-fold compared to 13,000-fold in fathead minnow). The concentration-response at the ovarian transcriptome level was nonmonotonic and violated assumptions that underlie proposed methods for estimating hazard thresholds from transcriptomic results. However, the nonmonotonic profile was consistent among species and there were nominal similarities in the functions associated with the differentially expressed genes, suggesting potential activation of common pathway perturbation motifs in both species. Overall, the results provide an effective case study for considering the potential application of ecotoxicogenomics to ecological risk assessments and provide novel comparative data regarding effects of BPA in fish.

The fish early life-stage (FELS) test guideline (OECD 210 or OCSPP 850.1400) is the most frequently used bioassay for predicting chronic fish toxicity and supporting aquatic ecological risk assessments around the world. For each chemical, the FELS test requires a minimum of 360 fish and 1 to 3 months from test initiation to termination. Although valuable for predicting fish full life-cycle toxicity, FELS tests are labor and resource intensive and, due to an emphasis on apical endpoints, provide little to no information about chemical mode of action. Therefore, the development and implementation of alternative testing strategies for screening and prioritizing chemicals has the potential to reduce the cost and number of animals required for estimating FELS toxicity and, at the same time, provides insights into mechanisms of toxicity. Using three reference chemicals with well-established yet distinct adverse outcome pathways (AOPs) in early life stages of fish, we proposed FELS-specific AOPs as conceptual frameworks for identifying useful chemical screening and prioritization strategies. The reference chemicals selected as case studies were a cardiotoxic aryl hydrocarbon receptor agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin), neurotoxic acetylcholinesterase inhibitor (chlorpyrifos), and narcotic surfactant (linear alkylbenzene sulfonate). Using qualitative descriptions for each chemical during early fish development, we developed generalized AOPs and, based on these examples, proposed a three-tiered testing strategy for screening and prioritizing chemicals for FELS testing. Linked with biologically based concentration-response models, a tiered testing strategy may help reduce the reliance on long-term and costly FELS tests required for assessing the hazard of thousands of chemicals currently in commerce.

In recent years, therapeutic approaches including cytoreductive surgery followed by intraperitoneal chemotherapy have proven effective in peritoneal carcinomatosis of colorectal origin. If cytology is to be used to include patients in aggressive treatment regimens, it is necessary to evaluate its performance, particularly in terms of specificity. The aim of this study was to assess interobserver agreement for the detection of intraperitoneal free cancer cells (IFCCs) in patients with non-gynaecological adenocarcinomas.Over a 5-year period, 1223 patients were recruited in 19 French surgery departments. Peritoneal samples were examined in 14 dispersed pathology laboratories. Giemsa-stained slides were sent to a control reader blind to the previous diagnosis. Discordant cases, concordant positive results and a random selection of negative concordant cases were reviewed by a panel of seven cytopathologists. The 'final diagnosis' was that of the consensus meetings but took into account locally-processed slides.Gathering dubious cases with negative results, a 95.6% concordance was achieved between local readers and the control reader. IFCCs were ascertained by the panel in 85 cases (7.0%). Eight of 873 colorectal cancers cases viewed locally were falsely positive (0.9%). Radiotherapy and neoadjuvant therapy had no impact on the false-positive rate as assessed by final validation by the panel (P0.05). Samples initially considered as dubious were reclassified as negative by the panel in 24 of 25 cases (96.0%).The panel consensus allowed reclassification of most dubious/equivocal peritoneal cytology cases, whereas clearcut distinction between benign and malignant cases was correctly achieved in almost all cases.

The hypothalamus–pituitary–gonadal (HPG) axis plays a central role in the maintenance of homeostasis and disruptions in its function can have important implications for reproduction and other critical biological processes. A number of compounds found in aquatic environments are known to affect the HPG axis. In the present study, we used two-dimensional electrophoresis to investigate the proteome of female and male zebrafish brain after 96 h exposure to the fungicide prochloraz. Prochloraz has known effects on a number of key HPG molecules, including antagonism of Cyp17 and Cyp19 (aromatase). Twenty-eight proteins were shown to be differentially expressed in the brains of females and 22 in males. Proteins were identified using LC–MS/MS and identities were examined relative to brain function in the context of changing steroid hormone levels. There was little overlap between sexes in proteins exhibiting differential expression. Proteins with known roles in metabolism, learning, neuroprotection, and calcium regulation were determined to be differentially regulated. Relationships between identified proteins were also examined using Ingenuity Pathway Analysis, and females were shown to exhibit enrichment of several metabolic pathways. We used differentially expressed proteins to establish a putative classifier consisting of three proteins that was able to discriminate prochloraz-exposed from control females. Putatively impacted brain functions and specific protein changes that were observed have the potential to be generalized to other that similarly impact steroid hormone levels.

Certain endocrine-active toxicants have been reported to completely sex reverse both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species, resulting in a phenotype indistinguishable from unaffected individuals. Detection of low-level sex reversal often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with predominantly genetic sex determination and cryptic/homomorphic sex chromosomes. Here we describe a method for determining the genetic sex in the commonly used ecotoxicological model, the fathead minnow (Pimephales promelas). Analysis of amplified fragment length polymorphisms (AFLP) in a spawn of minnows resulted in detection of 10 sex-linked AFLPs, which were isolated and sequenced. No recombination events were observed with any sex-linked AFLP in the animals examined (n=112). A polymerase chain reaction (PCR) method was then developed that determined the presence of one of these sex-linked polymorphisms for utilization in routine toxicological testing. Analyses of additional spawns from our in-house culture indicate that fathead minnows utilize a XY sex determination strategy and confirm that these markers can be used to genotype sex; however, this method is currently limited to use in laboratory studies in which breeders possess a defined genetic makeup. The genotyping method described herein can be incorporated into endocrine toxicity assays that examine the effects of chemicals on gonad differentiation.

© 2011 Li et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background - Endocrine disrupting chemicals (e.g., estrogens, androgens and their mimics) are known to affect reproduction in fish. 17α-ethynylestradiol is a synthetic estrogen used in birth control pills. 17β-trenbolone is a relatively stable metabolite of trenbolone acetate, a synthetic androgen used as a growth promoter in livestock. Both 17α-ethynylestradiol and 17β-trenbolone have been found in the aquatic environment and affect fish reproduction. In this study, we developed a physiologically-based computational model for female fathead minnows (FHM, Pimephales promelas), a small fish species used in ecotoxicology, to simulate how estrogens (i.e., 17α-ethynylestradiol) or androgens (i.e., 17β-trenbolone) affect reproductive endpoints such as plasma concentrations of steroid hormones (e.g., 17β-estradiol and testosterone) and vitellogenin (a precursor to egg yolk proteins). Results - Using Markov Chain Monte Carlo simulations, the model was calibrated with data from unexposed, 17α-ethynylestradiol-exposed, and 17β-trenbolone-exposed FHMs. Four Markov chains were simulated, and the chains for each calibrated model parameter (26 in total) converged within 20,000 iterations. With the converged parameter values, we evaluated the model's predictive ability by simulating a variety of independent experimental data. The model predictions agreed with the experimental data well. Conclusions - The physiologically-based computational model represents the hypothalamic-pituitary-gonadal axis in adult female FHM robustly. The model is useful to estimate how estrogens (e.g., 17α-ethynylestradiol) or androgens (e.g., 17β-trenbolone) affect plasma concentrations of 17β-estradiol, testosterone and vitellogenin, which are important determinants of fecundity in fish. The Medical Research Foundation of Oregon, U.S. Environmental Protection Agency, and the National Center for Computational Toxicology of the EPA Office of Research and Development.

We evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for identifying aeromonads with an extraction procedure. Genus-level accuracy was 100%. Compared to rpoB gene sequencing, species-level accuracy was 90.6% (29/32) for type and reference strains and 91.4% for a collection of 139 clinical and environmental isolates, making this system one of the most accurate and rapid methods for phenotypic identification. The reliability of this technique was very promising, although some improvements in database composition, taxonomy, and discriminatory power are needed.

The impact of exposure by water to a model androgen, 17β-trenbolone (TRB), was assessed in fathead minnows using an integrated molecular approach. This included classical measures of endocrine exposure such as impacts on testosterone (T), 17β-estradiol (E2), and vitellogenin (VTG) concentrations in plasma, as well as determination of effects on the hepatic metabolome using proton nuclear magnetic resonance spectroscopy. In addition, the rates of production of T and E2 in ovary explants were measured, as were changes in a number of ovarian gene transcripts hypothesized to be relevant to androgen exposure. A temporally intensive 16-d test design was used to assess responses both during and after the TRB exposure (i.e., depuration/recovery). This strategy revealed time-dependent responses in females (little impact was seen in the males), in which changes in T and E2 production in the ovary, as well as levels in plasma, declined rapidly (within 1 d), followed shortly by a return to control levels. Gene expression measurements revealed dynamic control of transcript levels in the ovary and suggested potential mechanisms for compensation during the exposure phase of the test. Proton nuclear magnetic resonance spectroscopy revealed a number of hepatic metabolite changes that exhibited strong time and dose dependence. Furthermore, TRB appeared to induce the hepatic metabolome of females to become more like that of males at both high test concentrations of TRB (472 ng/L) and more environmentally relevant levels (33 ng/L).

To study mechanisms underlying generalized effects of 3β hydroxysteroid dehydrogenase (HSD3B) inhibition, reproductively mature zebrafish (Danio rerio) were exposed to trilostane at two dosages for 24, 48, or 96 h and their gonadal RNA samples profiled with Agilent zebrafish microarrays. Trilostane had substantial impact on the transcriptional dynamics of zebrafish, as reflected by a number of differentially expressed genes (DEGs) including transcription factors (TFs), altered TF networks, signaling pathways, and Gene Ontology (GO) biological processes. Changes in gene expression between a treatment and its control were mostly moderate, ranging from 1.3 to 2.0 fold. Expression of genes coding for HSD3B and many of its transcriptional regulators remained unchanged, suggesting transcriptional up-regulation is not a primary compensatory mechanism for HSD3B enzyme inhibition. While some trilostane-responsive TFs appear to share cellular functions linked to endocrine disruption, there are also many other DEGs not directly linked to steroidogenesis. Of the 65 significant TF networks, little similarity, and therefore little cross-talk, existed between them and the hypothalamic-pituitary-gonadal (HPG) axis. The most enriched GO biological processes are regulations of transcription, phosphorylation, and protein kinase activity. Most of the impacted TFs and TF networks are involved in cellular proliferation, differentiation, migration, and apoptosis. While these functions are fairly broad, their underlying TF networks may be useful to development of generalized toxicological screening methods. These findings suggest that trilostane-induced effects on fish endocrine functions are not confined to the HPG-axis alone. Its impact on corticosteroid synthesis could also have contributed to some system wide transcriptional changes in zebrafish observed in this study.

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.

Various assays with adult fish have been developed to identify potential endocrine-disrupting chemicals (EDCs) which may cause toxicity via alterations in the hypothalamic-pituitary-gonadal (HPG) axis. These assays can be sensitive and highly diagnostic for key mechanisms such as agonism of the estrogen and androgen receptors (ERs, ARs) and inhibition of steroid synthesis. However, most of the tests do not unambiguously identify AR antagonists. The purpose of this work was to explore the utility of a mixture test design with the fathead minnow (Pimephales promelas) for detecting different classes of EDCs including AR antagonists. Adults of both sexes were exposed via the water to EDCs with diverse mechanisms of action in the absence or presence of 17beta-trenbolone (TB), a potent AR agonist which masculinizes female fathead minnows. Similar to previous studies with the model AR antagonists flutamide and vinclozolin, exposure of females to the AR antagonist cyproterone acetate in the presence of TB decreased expression of an easily-observed masculinization response, nuptial tubercle formation. Mixture studies with TB and the model ER agonists, 17alpha-ethinylestradiol and bisphenol A, also showed inhibition of tubercle formation in the females, but unlike the AR antagonists, the estrogens markedly induced synthesis of vitellogenin (VTG: egg yolk protein), particularly in males. The ER agonists also offset TB-induced depressions in plasma VTG concentrations in female fish. Additional mixture experiments were conducted with TB and triclocarban, an anti-microbial reported to enhance AR-mediated responses, or ammonia, a "negative control" with no known direct effects on HPG function. Neither chemical affected VTG status in males or females in the absence or presence of TB; however, both slightly enhanced TB-induced tubercle formation in females. Based on studies described herein and elsewhere with the fathead minnow, a TB co-exposure assay appears to be an effective approach for clearly identifying AR antagonists as well as potential EDCs with other relevant mechanisms of action.

Synthetic and natural steroidal androgens and estrogens and many other non-steroidal endocrine-active compounds commonly occur as complex mixtures in aquatic environments. It is important to understand the potential interactive effects of these mixtures to properly assess their risk. Estrogen receptor agonists exhibit additivity in mixtures when tested in vivo and in vitro. Little is known, however, concerning possible mixture interactions of androgen receptor agonists. In these studies we used the MDA-kb2 cell line, a human breast cancer cell line with endogenous androgen receptors and a stably transfected luciferase reporter gene construct to quantify the androgenic activity of seven natural and synthetic androgens: 17β-trenbolone, dihydrotestosterone, methyltestosterone, testosterone, trendione, 17α-trenbolone, and androstenedione. We tested combinations of these androgens and compared the observed activity to expected androgenic activity based on a concentration addition model. Our analyses support the hypothesis that androgen receptor agonists cause additive responses in a mixture. Binary mixtures of 17β-trenbolone with 17β-estradiol or triclocarban (an anti-microbial found in the environment) were also tested. 17β-Estradiol induced androgenic activity, but only at concentrations 600-fold greater than those found in the environment. Triclocarban enhanced the activity of 17β-trenbolone. Additionally, three anti-androgens were each paired with three androgens of varying potencies. The relative potencies of the antagonists were a vinclozolin metabolite (M2) > procymidone > prochloraz regardless of the androgen used. The results of our studies demonstrate the potential utility of the androgen-responsive MDA-kb2 cell line for quantifying the activity of mixtures of endocrine-active chemicals in complex wastes such as municipal effluents and feedlot discharges. Environ. Toxicol. Chem. 2010;29:1367–1376. © 2010 SETAC

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms that maintain a dynamic homeostasis in the face of changing environmental conditions, including exposure to chemicals. We assessed the effects of prochloraz on HPG axis function in adult fathead minnows (Pimephales promelas) at multiple sampling times during 8-day exposure and 8-day depuration/recovery phases. Consistent with one mechanism of action of prochloraz, inhibition of cytochrome P450 (CYP) 19 aromatase activity, the fungicide depressed ex vivo ovarian production and plasma concentrations of 17beta-estradiol (E2) in female fish. At a prochloraz water concentration of 30 microg/l, inhibitory effects on E2 production were transitory and did not persist during the 8-day exposure phase. At 300 microg/l prochloraz, inhibition of E2 production was evident throughout the 8-day exposure but steroid titers recovered within 1 day of cessation of exposure. Compensation or recovery of steroid production in prochloraz-exposed females was accompanied by upregulation of several ovarian genes associated with steroidogenesis, including cyp19a1a, cyp17 (hydroxylase/lyase), cyp11a (cholesterol side-chain cleavage), and follicle-stimulating hormone receptor. In male fathead minnows, the 8-day prochloraz exposure decreased testosterone (T) production, possibly through inhibition of CYP17. However, as for E2 in females, ex vivo testicular production and plasma concentrations of T recovered within 1 day of stopping exposure. Steroidogenic genes upregulated in testis included cyp17 and cyp11a. These studies demonstrate the adaptability of the HPG axis to chemical stress and highlight the need to consider the dynamic nature of the system when developing approaches to assess potential risks of endocrine-active chemicals.

Trenbolone, an anabolic androgen, and flutamide, an antiandrogen, are prototypical model compounds for agonism and antagonism of the androgen receptor. We hypothesized that 48 h exposures of female fathead minnows (Pimephales promelas) to environmentally relevant concentrations of these chemicals would alter genes regulated by the androgen receptor and that a mixture of the two compounds would block the effects. Gene expression in the ovaries was analyzed using a fathead minnow-specific 22,000-gene microarray. Flutamide altered abouttwicethe number of genes astrenbolone, most of which appeared to be through pathways not associated with the androgen receptor. A group of 70 genes, of which we could identify 37, were reciprocally regulated by trenbolone and flutamide. These are candidates for specific biomarkers for androgen receptor mediated gene expression. Four genes stand out as specifically related to reproduction: sperm associated antigen 8 (SPAG8), CASP8 and FADD-like apoptosis regulator (CFLAR), corticotropin releasing hormone (CRH), and 3beta-hydroxysteroid dehydrogenases (3beta-HSD). Three notable transcriptional regulators including myelocytomatosis viral oncogene homologue (MYC), Yin Yang 1 (YY1), and interferon regulator factor 1 (IRF1) may function as early molecular switches to control phenotypic changes in ovary tissue architecture and function in response to androgen or antiandrogen exposure.

Neurotransmitters such as dopamine play an important role in regulating fish reproduction. However, the potential for neuroendocrine active chemicals to disrupt fish reproduction has not been well studied, despite emerging evidence of their discharge into aquatic environments. This study is the first to apply the fathead minnow 21 d reproduction assay developed for the US Endocrine Disruptor Screening Program to evaluate the reproductive toxicity of a model neuroendocrine active chemical, the dopamine 2 receptor antagonist, haloperidol. Continuous exposure to up to 20 imcrog haloperidol/L had no significant effects on fathead minnow fecundity, secondary sex characteristics, gonad histology, or plasma steroid and vitellogenin concentrations. The only significant effect observed was an increase in gonadotropin-releasing hormone (cGnRH) transcripts in the male brain. Results suggest that non-lethal concentrations of haloperidol do not directly impair fish reproduction. Potential effects of haloperidol on reproductive behaviors and gene expression were examined in a companion study.

Estrogenic chemicals in the aquatic environment have been shown to cause a variety of reproductive anomalies in fish including full sex reversal, intersex, and altered population sex ratios. Two estrogens found in the aquatic environment, 17alpha-ethinylestradiol (EE(2)) and 17beta-estradiol (E(2)), have been measured in wastewater treatment effluents and have been shown to cause adverse effects in fish. To further our understanding of how estrogen exposure affects reproductive endpoints in the male fathead minnow (FHM, Pimephales promelas), a physiologically based computational model was developed of the hypothalamic-pituitary-gonadal (HPG) axis. Apical reproductive endpoints in the model include plasma steroid hormone and vitellogenin concentrations. Using Markov chain Monte Carlo simulation, the model was calibrated with data from unexposed FHM, and FHM exposed to EE(2) and E(2). Independent experimental data sets were used to evaluate model predictions. We found good agreement between our model predictions and a variety of measured reproductive endpoints, although the model underpredicts unexposed FHM reproductive endpoint variances, and overpredicts variances in estrogen-exposed FHM. We conclude that this model provides a robust representation of the HPG axis in male FHM.

In a recent report, Toxicity Testing in the 21st Century, the National Research Council Committee on Toxicity Testing and Assessment of Environmental Agents (2007) proposed that improved scientific understanding of toxicity pathways was central to the expanded use of predictive, pathway-based bioassays in risk assessment. Toxicity pathways can be viewed as the series of biological changes, spanning across multiple levels of biological organization, that lead from some molecular initiating event (perturbation) to an adverse outcome. A major challenge associated with dose–response modeling and extrapolation from laboratory to real-world conditions has been to understand under what conditions an organism may compensate for, or recover from, a given perturbation and under what conditions the perturbation will lead to an adverse outcome (Andersen et al. 2005). Thus, in developing useful predictive models of toxicity, we need to understand not only the direct effects of a chemical and how they translate into adverse effect, but also the potential mechanisms for compensation and recovery and how they may intersect with other biological pathways and processes. Previous studies with the fathead minnow (Pimephales promelas) have suggested potential compensatory responses to the direct effects of chemicals whose primary mode of action was inhibition of one or more enzymes involved in steroid biosynthesis. For example, the chemical fadrozole (FAD) inhibits aromatase, the enzyme that catalyzes the rate-limiting conversion of testosterone (T) to 17β-estradiol (E2) (Miller 1988). Villeneuve et al. (2006) observed significant, concentration-dependent up-regulation of transcripts coding for the ovarian isoform of aromatase (CYP19A) in female fathead minnows exposed to FAD for 7 days. The increased CYP19A gene expression was associated with an inverted U-shaped concentration–response profile for ovary aromatase activity. Although that study did not examine effects on plasma E2 concentrations, it was noted that the responses would favor increased synthesis of E2 to potentially offset the effect of FAD on aromatase. In another study, Ankley et al. (2007) exposed fathead minnows to the steroidogenesis inhibitor ketoconazole for 21 days. Testosterone production by testis or ovary tissue collected from ketoconazole-exposed fish was significantly reduced compared with control fish. However, there was significant up-regulation of genes coding for cytochrome P450 cholesterol side-chain cleavage (P450scc, CYP11A) and cytochrome P450 c17αhydroxylase, 17,20-lyase, and concentration-dependent proliferation of steroid-producing interstitial cells in the testis of exposed males. As a result, plasma T and E2 concentrations in exposed fish were similar to those of controls, despite the decreased rate of steroid production per unit mass of tissue, suggesting a compensatory response to ketoconazole (Ankley et al. 2007). Both the FAD and ketocona zole studies suggest that fish have the capacity to adapt to and potentially recover from the direct effects of steroidogenesis inhibitors. Aromatase is a key steroidogenic enzyme shown to be subject to inhibition, at least in vitro, by a variety of chemicals present in the environment, including certain pesticides, organochlorines, and organotins (Sanderson 2006). The aim of this study was to develop a more comprehensive understanding of molecular and biochemical responses of fathead minnows to aromatase inhibition, including direct effects, compensation, and/or recovery. We examined a time course of selected gene expression and biochemical responses over the course of an 8-day waterborne exposure to two concentrations of FAD, followed by an 8-day recovery period in control water. The experiment was designed to test several hypotheses: FAD would elicit direct effects consistent with its presumptive mode of action Over time, there would be compensatory molecular responses in females, consistent with an attempt to increase E2 synthesis Compensatory effects at the molecular level would correspond to changes in circulating E2 and/or rates of ex vivo E2 production Effects would be time and concentration dependent There would be recovery after cessation of FAD exposure. Although FAD is a drug with no direct environmental relevance, its specificity makes it a useful model chemical for studying this mode of action. Results of this study provide an improved understanding of the dynamics of biological response to this chemical, and its removal. This knowledge will contribute to formulation of a robust, biologically based toxicity pathway model for the effects of estradiol synthesis inhibitors.

An in vitro ethoxyresorufin O-deethylase (EROD) assay was used to study the ability of individual polycyclic aromatic hydrocarbons (PAHs) and mixtures of PAHs to induce Ah receptor (AhR) mediated cytochrome P4501A activity in PLHC-1 fish hepatoma cells. The purpose was to identify the most potent inducers from a set of thirteen separate PAHs and describe interactions occurring in complex mixtures of these PAHs. Where possible, potency was expressed in terms of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TCDD-EQ) by normalizing the PAH results to a TCDD standard curve. The most potent inducers were benzo(k)fluoranthene > benzo(a)pyrene {approx} benzo(b)fluoranthene > chrysene {approx} benzo(a)anthracene. At equal concentrations, these PAHs yielded potencies of 1670, 940, 655, 255, and 185 pg TCDD-EQ/g, respectively. Analysis of various mixtures of the thirteen PAHs suggested that complex interactions may be occurring.

Coordinate production of interleukin-1 beta (IL-1 beta) and granulocyte macrophage-colony stimulating factor (GM-CSF) or IL-6 by the blast cells of acute myeloblastic leukemia (AML) and normal peripheral blood leukocytes have been previously reported (van der Shoot et al.: Blood 74:2081-2087, 1989; Bradbury et al.: Leukemia 4:44-47 1990a, British Journal of Haematology 16:(in press), 1990b; Rodriguez-Cimadevilla et al.: Blood 76:1481-1489, 1990; Schindler et al.: Blood 75:40-47, 1990). In the present study, we show that IL-6 production by AML blasts is up-regulated by endogenously produced IL-1 beta. Neutralization of the endogenous source of IL-1 results in a significant decrease in IL-6 production, as determined by ELISA. Conversely, exposure of AML blasts to IL-1 alpha results in a significant increase in IL-6 production in 10 of 16 patient samples. Antibodies against IL-1 alpha and -beta also cause a drastic decrease in IL-6 and GM-CSF gene expression by the cells, suggesting that cytokine gene expression in AML blasts is driven, at least in part, by endogenous IL-1. The biologic significance of IL-6 production in culture of AML blasts has been addressed using a neutralizing antibody against IL-6. Our data indicate that IL-6 is important for the survival of clonogenic blasts in culture. In contrast, the survival of the total population of blasts is IL-6-independent, as assessed by the integrity of cellular DNA, even in the presence of anti-IL-6. These observations are consistent with the view that AML blasts might be organized as a lineage, with comparable hierarchy as in normal hemopoiesis and, perhaps, increased heterogeneity despite a homogenous appearance (McCulloch and Till: Blood Cells 7:63-77, 1981; Buick and McCulloch: Control of Animal Cell Proliferation. Academic Press, New York, vol. 1, pp. 25-57, 1985). Buick and McCulloch have identified a subpopulation of AML clonogenic cells with stem-cell-like properties, and suggested that the majority of blasts may have undergone a determination-like step. Our data indicate a marked difference in IL-6 requirement for cell survival between precursors and the majority of blasts, suggesting that IL-6 responsiveness may decrease following a determination-like event, i.e., the reduction in proliferative capacity.

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 microg/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 microg/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.

Knowledge of possible toxic mechanisms (or modes) of action (MOA) of chemicals can provide valuable insights as to appropriate methods for assessing exposure and effects, thereby reducing uncertainties related to extrapolation across species, endpoints and chemical structure. However, MOA-based testing seldom has been used for assessing the ecological risk of chemicals. This is in part because past regulatory mandates have focused more on adverse effects of chemicals (reductions in survival, growth or reproduction) than the pathways through which these effects are elicited. A recent departure from this involves endocrine-disrupting chemicals (EDCs), where there is a need to understand both MOA and adverse outcomes. To achieve this understanding, advances in predictive approaches are required whereby mechanistic changes caused by chemicals at the molecular level can be translated into apical responses meaningful to ecological risk assessment. In this paper we provide an overview and illustrative results from a large, integrated project that assesses the effects of EDCs on two small fish models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). For this work a systems-based approach is being used to delineate toxicity pathways for 12 model EDCs with different known or hypothesized toxic MOA. The studies employ a combination of state-of-the-art genomic (transcriptomic, proteomic, metabolomic), bioinformatic and modeling approaches, in conjunction with whole animal testing, to develop response linkages across biological levels of organization. This understanding forms the basis for predictive approaches for species, endpoint and chemical extrapolation. Although our project is focused specifically on EDCs in fish, we believe that the basic conceptual approach has utility for systematically assessing exposure and effects of chemicals with other MOA across a variety of biological systems.

Ketoconazole is a fungicidal drug that inhibits function of cytochrome P450s in the synthesis of steroids. To examine if inhibition of P450 function affects gene expression in a dynamic manner, we conducted in vitro exposures of ovary tissue from fathead minnows (Pimephales promelas) to 0.5 μM ketoconazole to investigate effects on steroid production and gene expression over time. Expression of four key steroidogenesis genes was examined at 1, 6, and 12 h of exposure. 11β- and 20β-hydroxysteroid dehydrogenases were down regulated at 1 h and Cytochrome P450 17 was down-regulated at 12 h, consistent with the absence of steroid production. In contrast, cytochrome P450 19A was up-regulated at 6 h, indicating feedback regulation. Microarray analysis of 12 h exposures indicated enrichment of biological processes involved in neurotransmitter secretion, lymphocyte cell activation, sodium ion transport, and embryonic development. These data suggest that, with the exception of cytochrome P450 19A, these steroid metabolic genes are regulated in a feed forward manner and that the effects of ketoconazole may be broader than anticipated based on the mechanism of action alone.

A number of environmental contaminants and plant flavonoid compounds have been shown to inhibit the activity of 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD). Because 3beta-HSD plays a critical role in steroid hormone synthesis, inhibition of 3beta-HSD represents a potentially important mode of endocrine disruption that may cause reproductive dysfunction in fish or other vertebrates. The objective of this study was to test the hypothesis that exposure to the model 3beta-HSD inhibitor, trilostane, would adversely affect reproductive success of the fathead minnow (Pimephales promelas). Results of in vitro experiments with fathead minnow ovary tissue demonstrated that trilostane inhibited 17beta-estradiol (E2) production in a concentration- and time-dependent manner, and that the effect was eliminated by providing a substrate (progesterone) that does not require 3beta-HSD activity for conversion to E2. Exposure of fish to trilostane caused a significant reduction in spawning frequency and reduced cumulative egg production over the course of the 21-day test. In females, exposure to 1500 mug trilostane/l reduced plasma vitellogenin concentrations, but did not cause significant histological alterations. In males, average trilostane concentrations as low as 50 mug/l significantly increased testis mass and gonadal somatic index. Trilostane exposure did not influence the abundance of mRNA transcripts coding for 3beta-HSD or other steroidogenesis-regulating proteins in males or females. As a whole, results of this study support the hypothesis that 3beta-HSD inhibition can cause reproductive dysfunction in fish, but did not yield a clear profile of responses at multiple levels of biological organization that could be used to diagnose this mode of action.

Concentration and/or production of sex steroids such as 17beta-estradiol (E2) and testosterone (T) in fish have commonly been measured in field studies concerned with endocrine-active chemicals. There is a reasonable mechanistic basis for using E2 or T as biomarkers, as chemicals can alter steroid production through both direct and indirect effects on the hypothalamic-pituitary-gonadal (HPG) axis. There is uncertainty, however, as to what changes in steroid status may mean relative to apical endpoints, such as reproduction, that directly affect population status. In this study, we analyzed data from fathead minnow (Pimephales promelas) reproduction studies in which decreases in fecundity were associated with depressed steroid production as a result of chemical exposure. Although the chemicals acted on the HPG axis through different mechanisms, reproductive effects appeared to be expressed through a common pathway, depression of vitellogenin production in females. Plasma concentrations of E2 or T in the females were significantly, positively correlated with fecundity. Linear regression models describing the relationship between E2 or T concentrations and relative fecundity were linked to a population model to predict population trajectories of fathead minnows exposed to chemicals that inhibit steroid production. For example, a population existing at carrying capacity and exposed to a chemical stressor(s) that causes a 50% decrease in E2 production was predicted to exhibit a 92% decrease in population size over a 5-year period. Results of our analysis illustrate a conceptual framework whereby a commonly measured biomarker, sex steroid status, could be linked to individual- and population-level effects in fish.

Acute myeloblastic leukemia (AML) blasts have been shown to produce a variety of cytokines in culture such as interleukin-1 (IL-1), IL-6, granulocyte-, macrophage-, and granulocyte-macrophage colony- stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF alpha). Using two sensitive and specific enzyme-linked immunosorbent assays for IL-1 beta and GM-CSF, we document in the present study that the production of the two cytokines by AML blasts in culture is coordinated. First, we observe a striking correlation between the levels of GM-CSF and IL-1 beta released by the cells. Thus, a high production of IL-1 beta is always concordant with a high production of GM-CSF and, conversely, low production of IL-1 beta is concordant with low levels of GM-CSF. Second, neutralization of intrinsic IL-1 using antibodies that are specific for IL-1 alpha and -1 beta suppresses the release of GM-CSF by the cells. Third, neutralization of the endogenous source of IL-1 also results in an abrogation of GM-CSF mRNA. Fourth, the production of both IL-1 beta and GM-CSF is up-regulated by exposing AML blasts to an exogenous source of IL-1, suggesting a positive regulation of autocrine growth factor production. Taken together, our results indicate that GM-CSF production by AML blasts is mediated by endogenously produced IL-1. Both IL-1 beta and -1 alpha are produced by AML blasts, although IL-1 beta appears to be more abundant. Spontaneous colony formation by AML blasts is abrogated by the addition of neutralizing antibodies against IL-1 beta and GM-CSF, whereas each antibody alone has little effect on blast proliferation. Taken together, our results are consistent with the view that the production of IL-1 beta by AML blasts supports autocrine growth in culture, through induction of CSFs or other cytokines that stimulate blast proliferation.

This paper summarizes the procedure and detail phase noise simulation result for a 38 GHz dual-loop push-push synthesizer, which requires low phase noise, high susceptibility of micro-phonic and phase hit performance over temperature. Push- Push dual loop synthesizer scheme has been used, one narrow band loop is designed for achieving low phase noise with small step size and the second loop is designed for a wide loop bandwidth for high susceptibility of micro-phonic and phase-hit over temperature. This paper shows a step-by-step simulation for a dual loop synthesizer. The simulation includes two push-push VCOs, one narrow band loop for the reference VCO, one wide band loop for the RF VCO, 10 MHz reference, PLL and other RF circuitry. The simulation and prototype testing results match well. The paper also highlights the typical test setups for phase hits and micro-phonics.

This study evaluated changes in the expression of steroidogenesis-related genes in male fathead minnows exposed to ketoconazole (KTC) or vinclozolin (VZ) for 21 days. The aim was to evaluate links between molecular changes and higher level outcomes after exposure to endocrine-active chemicals (EACs) with different modes of action. To aid our analysis and interpretation of EAC-related effects, we first examined variation in the relative abundance of steroidogenesis-related gene transcripts in the gonads of male and female fathead minnows as a function of age, gonad development, and spawning status, independent of EAC exposure. Gonadal expression of several genes varied with age and/or gonadal somatic index in either males or females. However, with the exception of aromatase, steroidogenesis-related gene expression did not vary with spawning status. Following the baseline experiments, expression of the selected genes in male fathead minnows exposed to KTC or VZ was evaluated in the context of effects observed at higher levels of organization. Exposure to KTC elicited changes in gene transcription that were consistent with an apparent compensatory response to the chemical's anticipated direct inhibition of steroidogenic enzyme activity. Exposure to VZ, an antiandrogen expected to indirectly impact steroidogenesis, increased pituitary expression of follicle-stimulating hormone beta-subunit as well as testis expression of 20beta-hydroxysteroid dehydrogenase and luteinizing hormone receptor transcripts. Results of this study contribute to ongoing research aimed at understanding responses of the teleost hypothalamic-pituitary-gonadal axis to different types of EACs and how changes in molecular endpoints translate into apical outcomes reflective of either adverse effect or compensation.

A challenge in the field of ecotoxicology is the linkage of alterations at molecular and biochemical levels of organization to adverse outcomes in individuals and populations. In the present study, a predictive relationship between plasma vitellogenin (VTG) concentration and fecundity in female fathead minnows (Pimephales promelas) was derived from 21-d laboratory toxicity tests with five chemicals (17beta-trenbolone, 17alpha-trenbolone, prochloraz, fenarimol, and fadrozole) that inhibit VTG production through different mechanisms. Because VTG is key to egg production in female oviparous animals, changes in the lipoprotein could, theoretically, serve as an indicator of reproductive success. Regression of fecundity versus VTG concentration from the various studies yielded a highly significant linear model (fecundity = -0.042 + 0.95 x VTG, p0.01, r2 = 0.88). This relationship was integrated into a population model to translate changes in VTG concentrations of female fathead minnows to alterations in population growth. The model predicted relatively profound effects on population size of fish experiencing moderate decreases in vitellogenesis. For example, a fathead minnow population at a carrying capacity exposed to a chemical stressor that causes a 25% decrease in VTG concentration in females from baseline values would exhibit a 34.6% projected decrease in size after two years of exposure and reach an equilibrium population size that was only 30.2% of the preexposed population. Overall, the current study provides an example of how changes in a biomarker (VTG concentration) can be quantitatively translated into adverse effects at the individual and population levels.

As potential biomarkers, gene classifiers are gene expression signatures or patterns capable of distinguishing biological samples belonging to different classes or conditions. This is the second of two papers on profiling gene expression in zebrafish (Danio rerio) treated with endocrine-disrupting chemicals of different modes of action, with a focus on comparative analysis of microarray data for gene classifier discovery. Various combinations of gene feature selection/class prediction algorithms were evaluated, with the use of microarray data organized by a chemical stressor or tissue type, for their accuracy in determining the class memberships of independent test samples. Two-way clustering of gene classifiers and treatment conditions offered another alternative to assess the performance of these potential biomarkers. Both gene feature selection methods and class prediction algorithms were shown to be important in identifying successful gene classifiers. The genetic algorithm and support vector machine yielded classifiers with the best prediction accuracy, regardless of sample size, nature of class prediction, and data complexity. A chemical stressor significantly altering the expression of a greater number of genes tended to generate gene classifiers with better performance. All combinations of gene feature selection/class prediction algorithms performed similarly well with data of high signal to noise ratio. Gene classifier discovery and application on the basis of individual sampling and sample data pooling, respectively, were found to enhance class predictions. Gene expression profiles of the top gene classifiers, identified from both microarray and quantitative polymerase chain reaction assays, displayed greater similarity between fadrozole and 17beta-trenbolone than either one to 17alpha-ethinylestradiol. These gene classifiers could serve as potential biomarkers of exposure to specific classes of endocrine disruptors.

This paper is devoted to the capacitance computing and characterization of interconnection crossing and patch used in multilevel package. The [C] matrix calculation is performed with scalar potential given in its integral form. Analytical formulas easy to use in CAD systems have been derived from numerical results using least square method. A good agreement has been achieved between simulations and experimental results.

This study investigates the effect of the synthesizer phase noise shape for a multi baud rate radio. The trade-off between the synthesizer loop bandwidth and the noise contribution in the communication system is presented. This work shows also the effect of the oscillator technology on the integrated phase noise performance.