Growth inhibition and stimulation of Shewanella oneidensis MR-1 by surfactants and calcium polysulfide.

Foam delivery technology (FDT) uses surfactant based foam to immobilize subsurface contaminants in situ. Where traditional approaches are impractical, FDT has the potential to overcome many of the technical challenges facing the remediation of contaminated deep vadose zone environments. However, little is known about the effects these reactive chemicals may have on microorganisms inhabiting the contaminated subsurface. In addition, there are currently no standard assays to assess microbial responses to subsurface remedial treatments while these agents are under development. The objective of this study was to develop a rapid laboratory assay to assess the potential growth inhibition and/or stimulation of microorganisms following exposure to candidate FDT components. Calcium polysulfide (CPS) and several surfactants (i.e. sodium laureth sulfate (SLES), sodium dodecyl sulfate (SDS), cocamidopropyl betaine (CAPB) and NINOL40-CO) have diverse chemistries and are candidate components of FDT. Shewanella oneidensis MR-1 cultures were exposed to a range of concentrations of these chemicals to determine the minimum bactericidal concentration (MBC) and the growth and viability potential of these components. Concentrations of SDS higher than 700 µM were toxic to S. oneidensis MR-1 growth over the course of four days of exposure. The relative acute toxicity order for these compounds was SDS >> CPS >> NINOL 40-CO>SLES≥CAPB. Dose dependent growth decreases (20-100mM) were observed in the CAPB and SLES treated cultures and both CPS and NINOL 40-CO were toxic at all concentrations tested (1.45-7.25 mM CPS). Both SLES (20-100mM) and SDS at lower concentrations (20-500 µM) were stimulatory to S. oneidensis MR-1 indicating a capacity to be used as a carbon source. These studies also identified potentially key component characteristics, such as precipitate formation and oxygen availability, which may prove valuable in assessing the response of subsurface microorganisms. This benchtop system provides a capability to assess adverse microbial-remediation responses and contributes to the development of in situ remedial chemistries before they are deployed in the field.

Spillway-induced salmon head injury triggers the generation of brain alphaII-spectrin breakdown product biomarkers similar to mammalian traumatic brain injury.

Recent advances in biomedical research have resulted in the development of specific biomarkers for diagnostic testing of disease condition or physiological risk. Of specific interest are alphaII-spectrin breakdown products (SBDPs), which are produced by proteolytic events in traumatic brain injury and have been used as biomarkers to predict the severity of injury in humans and other mammalian brain injury models. This study describes and demonstrates the successful use of antibody-based mammalian SBDP biomarkers to detect head injury in migrating juvenile Chinook salmon (Oncorhynchus tshawytscha) that have been injured during passage through high-energy hydraulic environments present in spillways under different operational configurations. Mortality and injury assessment techniques currently measure only near-term direct mortality and easily observable acute injury. Injury-based biomarkers may serve as a quantitative indicator of subacute physical injury and recovery, and aid hydropower operators in evaluation of safest passage configuration and operation actions for migrating juvenile salmonids. We describe a novel application of SBDP biomarkers for head injury for migrating salmon. To our knowledge, this is the first documented cross-over use of a human molecular biomarker in a wildlife and operational risk management scenario.

Investigating freshwater periphyton community response to uranium with phospholipid fatty acid and denaturing gradient gel electrophoresis analyses.

Periphyton communities can be used as monitors of ecosystem health and as indicators of contamination in lotic systems. Measures of biomass, community structure, and genetic diversity were used to investigate impacts of uranium (U) exposure on periphyton. Laboratory exposures of periphyton in river water amended with 238U were performed for 5 days, followed by 2 days of U depuration in unamended river water. Productivity as measured by biomass was not affected by concentrations up to 100 microg238U L(-1). Phospholipid fatty acid (PLFA) profiles and denaturing gradient gel electrophoresis (DGGE) banding patterns revealed no changes in community or genetic structure related to U exposure. We suggest that the periphyton community as a whole was not significantly impacted by exposures of 238U up to a concentration of 100 microgL(-1). These findings have significance for the assessment and prediction of U impacts on aquatic ecosystems.

Development and validation of a 2,000-gene microarray for the fathead minnow (Pimephales promelas).

Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17beta-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17beta-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol's expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species.

Transcription of key genes regulating gonadal steroidogenesis in control and ketoconazole- or vinclozolin-exposed fathead minnows.

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 graphical systems model to facilitate hypothesis-driven ecotoxicogenomics research on the teleost brain-pituitary-gonadal axis.

Graphical systems models are powerful tools that can help facilitate hypothesis-driven ecotoxicogenomic research and aid in mechanistic interpretation of results. This paper describes a novel graphical model of the teleost brain-pituitary-gonadal (BPG) axis designed for ecotoxicogenomics research on endocrine-disrupting chemicals using small fish models. The model incorporates six compartments representing the major organs involved in the fish reproductive axis and depicts the interactions of over 105 proteins and 40 simple molecules, transcriptional regulation of 25 genes, and over 300 different reactions/ processes. Application of the model is illustrated in the context of a study examining effects of the competitive aromatase inhibitor, fadrozole, on gene expression in gonad, brain, and liver tissue of fathead minnows. Changes in mRNA transcript abundance were measured using a fathead minnow oligonucleotide microarray and quantitative real-time polymerase chain reaction. Gene expression changes observed in the ovaries of females exposed to 6.3 microg fadrozole/L for7 d were functionally consistent with fadrozole's mechanism of action, and expected compensatory responses of the BPG axis to fadrozole's effects. Furthermore, microarray results helped identify additional elements (genes/ proteins) that could be included in the model to potentially increase its predictive capacity. With proper recognition of

Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin beta subunit mRNAs to support endocrine disruptor research.

Fathead minnows (Pimephales promelas) are a widely-used small fish model for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). Quantitative real-time PCR assays for measuring fathead minnow gonadotropin (GtH) beta subunit transcripts were developed and "baseline" transcript levels in pituitary tissue were examined over a range of age classes and spawning states. Among females, GtHbeta transcripts did not vary significantly with gonadal-somatic index or gonad stage. However, in males, follicle-stimulating hormone beta subunit transcripts decreased significantly with increasing gonad stage, while mean luteinizing hormone beta subunit expression trended in the opposite direction. GtHbeta transcript levels measured in pituitaries from fish that had spawned within the preceding 24 h were not significantly different from those from fish that were 2-3 days post-spawn. Exposure to the fungicide ketoconazole, a known steroidogenesis inhibitor, for 21 days significantly affected the abundance of GtHbeta transcripts in pituitary tissue in males, but not females. This study provides critical data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression. Results of such experiments should facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.

Expression of two vitellogenin genes (vg1 and vg3) in fathead minnow (Pimephales promelas) liver in response to exposure to steroidal estrogens and androgens.

In this study, we describe the sequence for the fathead minnow (Pimephales promelas) vitellogenin 3 gene (vg3) and compare the responses of vg1 and vg3 following exposure to steroidal estrogens and androgens. The fathead minnow vg3 sequence is the second nucleotide sequence described in teleosts, following the original description of this isoform in zebrafish (Danio rerio). Following a brief water exposure (24h) to 2, 5, and 10 ng/L 17alpha-ethynylestradiol (EE2), both vg1and vg3 were upregulated in male liver. However, levels of vg3 induction were four orders of magnitude lower than levels of induction of vg1. Suppression of vg in female liver following exposure to 50 or 500 ng/L of the synthetic androgen 17beta-trenbolone occurs at similar magnitude for both vg1 and vg3 isoforms. The results of this study support the use of vg1 as an indicator of estrogenic exposure in male fish and indicate the potential for vg1 and /or vg3 for use as indicators of androgenic exposure.

Ecotoxicogenomics: linkages between exposure and effects in assessing risks of aquatic contaminants to fish.

Understanding the biological effects of exposures to chemicals in the environment relies on classical methods and emerging technologies in the areas of genomics, proteomics, and metabonomics. Linkages between the historical and newer toxicological tools are currently being developed in order to predict and assess risk. Being able to classify chemicals and other stressors based on effects they have at the molecular, tissue, and organismal levels helps define a systems biology approach to development of streamlined, cost-effective, and comprehensive testing approaches for evaluating environmental hazards. The challenges of the individual technologies and the combinations of tools for ecotoxicogenomics are discussed in application to aquatic toxicology with a particular emphasis on fish testing.

Evaluation of the model anti-androgen flutamide for assessing the mechanistic basis of responses to an androgen in the fathead minnow (Pimephales promelas).

In this study, we characterized the effects of flutamide, a model mammalian androgen receptor (AR) antagonist, on endocrine function in the fathead minnow (Pimephales promelas), a small fish species that is widely used for testing endocrine-disrupting chemicals (EDCs). Binding assays with whole cells transiently transfected with cloned fathead minnow AR indicated that flutamide binds competitively to the receptor. However, as is true in mammalian systems, a 2-hydroxylated metabolite of flutamide binds to the AR with a much higher affinity than the parent chemical. Mixture experiments with flutamide and the androgen 17beta-trenbolone demonstrated that the anti-androgen effectively blocked trenbolone-induced masculinization (nuptial tubercle production) of female fathead minnows, indicating antagonism of an AR receptor-mediated response in vivo. Conversely, reductions in vitellogenin in trenbolone-exposed females were not blocked by flutamide, suggesting that the vitellogenin response is not directly mediated through the AR. The results of these studies provide data demonstrating the validity of using the fathead minnow as a model species for detecting EDCs that exert toxicity through interactions with the AR.

Evolutionary origins of lymphocytes: ensembles of T cell and B cell transcriptional regulators in a cartilaginous fish.

The evolutionary origins of lymphocytes can be traced by phylogenetic comparisons of key features. Homologs of rearranging TCR and Ig (B cell receptor) genes are present in jawed vertebrates, but have not been identified in other animal groups. In contrast, most of the transcription factors that are essential for the development of mammalian T and B lymphocytes belong to multigene families that are represented by members in the majority of the metazoans, providing a potential bridge to prevertebrate ancestral roles. This work investigates the structure and regulation of homologs of specific transcription factors known to regulate mammalian T and B cell development in a representative of the earliest diverging jawed vertebrates, the clearnose skate (Raja eglanteria). Skate orthologs of mammalian GATA-3, GATA-1, EBF-1, Pax-5, Pax-6, Runx2, and Runx3 have been characterized. GATA-3, Pax-5, Runx3, EBF-1, Spi-C, and most members of the Ikaros family are shown throughout ontogeny to be 1) coregulated with TCR or Ig expression, and 2) coexpressed with each other in combinations that for the most part correspond to known mouse T and B cell patterns, supporting conservation of function. These results indicate that multiple components of the gene regulatory networks that operate in mammalian T cell and B cell development were present in the common ancestor of the mammals and the cartilaginous fish. However, certain factors relevant to the B lineage differ in their tissue-specific expression patterns from their mouse counterparts, suggesting expanded or divergent B lineage characteristics or tissue specificity in these animals.

The path from molecular indicators of exposure to describing dynamic biological systems in an aquatic organism: microarrays and the fathead minnow.

The extent to which humans and wildlife are exposed to toxicants is an important focus of environmental research. This work has been directed toward the development of molecular indicators diagnostic for exposure to various stressors in freshwater fish. Research includes the discovery of genes, indicative of environmental exposure, in the Agency's long-established aquatic toxicological organism, the fathead minnow (Pimephales promelas). Novel cDNAs and coding sequences will be used in DNA microarray analyses for pattern identification of stressor-specific, differentially up- and down-regulated genes. The methods currently used to discover genes in this organism, for which few annotated nucleic acid sequences exist, are cDNA subtraction libraries, differential display, exploiting PCR primers for known genes of other members of the family Cyprinidae and use of degenerate PCR primers designed from regions of moderate protein homology. Single or multiple genes noted as being differentially expressed in microarray analyses will then be used in separate studies to measure bioavailable stressors in the laboratory and field. These analyses will be accomplished by quantitative RT-PCR. Moving from analysis of single gene exposures to the global state of the transcriptome offers possibilities that those genes identified by DNA microarray analyses might be critical components of dynamic biological systems and networks, wherein chemical stressors exert toxic effects through various modes of action. Additionally, the ability to discriminate bioavailability of stressors in complex environmental mixtures, and correlation with adverse effects downstream from these early molecular events, presents challenging new ground to be broken in the area of risk assessment.

Terminal deoxynucleotidyl transferases from elasmobranchs reveal structural conservation within vertebrates.

The DNA polymerase (pol) X family is an ancient group of enzymes that function in DNA replication and repair (pol beta), translesion synthesis (pol lambda and pol micro) and terminal addition of non-templated nucleotides. This latter terminal deoxynucleotidyl transferase (TdT) activity performs the unique function of providing diversity at coding joins of immunoglobulin and T-cell receptor genes. The first isolated full-length TdT genes from shark and skate are reported here. Comparisons with the three-dimensional structure of mouse TdT indicate structural similarity with elasmobranch orthologues that supports both a template-independent mode of replication and a lack of strong nucleotide bias. The vertebrate TdTs appear more closely related to pol micro and fungal polymerases than to pol lambda and pol beta. Thus, unlike other molecules of adaptive immunity, TdT is a member of an ancient gene family with a clear gene phylogeny and a high degree of similarity, which implies the existence of TdT ancestors in jawless fishes and invertebrates.

Effects of angiotensin-converting enzyme inhibitor on delayed-onset doxorubicin-induced cardiotoxicity.

Childhood survivors of cancer who are treated with anthacycline chemotherapy, such as doxorubicin (DOX), can develop late-onset cardiomyopathy years after chemotherapy. The mechanism(s) for progression of anthracycline cardiotoxicity to late cardiomyopathy is unknown. Because angiotensin II has been implicated in the progression of other cardiomyopathies, this investigation was undertaken to determine whether treatment with an angiotensinconverting enzyme (ACE) inhibitor, lisinopril, reduces the time-dependent effects of doxorubicin on cardiac gene expression and myocellular apoptosis. A single dose of saline (control) or doxorubicin (DOX treated; 2 mg/kg iv) was administered to rabbits. Control and DOX-treated groups were also subgrouped to receive lisinopril and designated as lisinopril or DOX + lisinopril, respectively (1 mg/kg/d oral), for 10 wk. Histopathology, as determined at the light and ultrastructural level, was consistent with a reduced number of cardiomyocytes relative to interstitial cells in the left ventricle (LV) of the DOX-treated group compared with control and DOX + lisinopril groups. Gene expression of the pro-atrial naturetic peptide (ANP), quantified by steady-state messenger ribonucleic acid (mRNA) levels with reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blotting, increased approx 12-fold (n = 10; p < 0.05) in the LV of DOX-treated groups compared to control and DOX + lisinopril groups. Increased ANP mRNA expression following doxorubicin dosing was localized predominantly in ventricular myocytes by in situ hybridization. Deoxyribonucleic acid (DNA) fragmentation, determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling), was increased in both DOX-treated and DOX + lisinopril groups compared to the control group. Lisinopril prevented both late-onset increased ventricular ANP expression and subsequent DOX-induced myocyte loss. The authors speculate that these protective effects of lisinopril are related to reduced ANP expression and myocyte loss, the latter possibly mediated by effects on myocellular apoptosis.

17alpha-ethynylestradiol-induced vitellogenin gene transcription quantified in livers of adult males, larvae, and gills of fathead minnows (Pimephales promelas).

We have applied a method for quantifying relative levels of messenger RNA (mRNA) transcription to assess chemically induced gene expression in fathead minnows (Pimephales promelas). Synthetic oligonucleotides designed for the fathead minnow vitellogenin gene transcription product were used in a reverse transcription polymerase chain reaction (RT-PCR) protocol. This sensitive and rapid strategy detected vitellogenin gene transcription in livers of male fathead minnows exposed to concentrations as low as 2 ng/L of the endocrine-disrupting compound 17alpha-ethynylestradiol for 24 h. Surprisingly, vitellogenin transcription products also were detected in gill tissue and in 48-h-old posthatch fathead minnow larvae. Relative levels of vitellogenin gene induction among individuals were quantified in a single-step reaction (PCR multiplex) with 18S rRNA universal primers and Competimers concurrently with fathead minnow vitellogenin oligonucleotides. This quantitative approach will markedly enhance detection of the first cellular event of estrogenic exposure to aquatic ecosystems in both field and laboratory systems. Use of the model provides sensitivity of detection at a concentration below those that cause mortality or visible signs of stress in fish or other aquatic organisms. The model may also provide an in vivo screening method for estrogenlike endocrine-disrupting compounds.