Laura S. Inouye

Cloning, Analysis and Functional Annotation of Expressed Sequence tags from the Earthworm Eisenia fetida

BackgroundEisenia fetida, commonly known as red wiggler or compost worm, belongs to the Lumbricidae family of the Annelida phylum. Little is known about its genome sequence although it has been extensively used as a test organism in terrestrial ecotoxicology. In order to understand its gene expression response to environmental contaminants, we cloned 4032 cDNAs or expressed sequence tags (ESTs) from two E. fetida libraries enriched with genes responsive to ten ordnance related compounds using suppressive subtractive hybridization-PCR.ResultsA total of 3144 good quality ESTs (GenBank dbEST accession number EH669363–EH672369 and EL515444–EL515580) were obtained from the raw clone sequences after cleaning. Clustering analysis yielded 2231 unique sequences including 448 contigs (from 1361 ESTs) and 1783 singletons. Comparative genomic analysis showed that 743 or 33% of the unique sequences shared high similarity with existing genes in the GenBank nr database. Provisional function annotation assigned 830 Gene Ontology terms to 517 unique sequences based on their homology with the annotated genomes of four model organisms Drosophila melanogaster, Mus musculus, Saccharomyces cerevisiae, and Caenorhabditis elegans. Seven percent of the unique sequences were further mapped to 99 Kyoto Encyclopedia of Genes and Genomes pathways based on their matching Enzyme Commission numbers. All the information is stored and retrievable at a highly performed, web-based and user-friendly relational database called EST model database or ESTMD version 2.ConclusionThe ESTMD containing the sequence and annotation information of 4032 E. fetida ESTs is publicly accessible at http://mcbc.usm.edu/estmd/.

Transcriptomic analysis of RDX and TNT interactive sublethal effects in the earthworm Eisenia fetida

BackgroundExplosive compounds such as TNT and RDX are recalcitrant contaminants often found co-existing in the environment. In order to understand the joint effects of TNT and RDX on earthworms, an important ecological and bioindicator species at the molecular level, we sampled worms (Eisenia fetida) exposed singly or jointly to TNT (50 mg/kg soil) and RDX (30 mg/kg soil) for 28 days and profiled gene expression in an interwoven loop designed microarray experiment using a 4k-cDNA array. Lethality, growth and reproductive endpoints were measured.ResultsSublethal doses of TNT and RDX had no significant effects on the survival and growth of earthworms, but significantly reduced cocoon and juvenile counts. The mixture exhibited more pronounced reproductive toxicity than each single compound, suggesting an additive interaction between the two compounds. In comparison with the controls, we identified 321 differentially expressed transcripts in TNT treated worms, 32 in RDX treated worms, and only 6 in mixture treated worms. Of the 329 unique differentially expressed transcripts, 294 were affected only by TNT, 24 were common to both TNT and RDX treatments, and 3 were common to all treatments. The reduced effects on gene expression in the mixture exposure suggest that RDX might interact in an antagonistic manner with TNT at the gene expression level. The disagreement between gene expression and reproduction results may be attributed to sampling time, absence of known reproduction-related genes, and lack of functional information for many differentially expressed transcripts. A gene potentially related to reproduction (echinonectin) was significantly depressed in TNT or RDX exposed worms and may be linked to reduced fecundity.ConclusionsSublethal doses of TNT and RDX affected many biological pathways from innate immune response to oogenesis, leading to reduced reproduction without affecting survival and growth. A complex interaction between mixtures of RDX and TNT was observed at the gene expression level that requires further study of the dynamics of gene expression and reproductive activities in E. fetida. These efforts will be essential to gain an understanding of the additive reproductive toxicity between RDX and TNT.

Comparison of transcriptional responses in liver tissue and primary hepatocyte cell cultures after exposure to hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine.

BackgroundCell culture systems are useful in studying toxicological effects of chemicals such as Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), however little is known as to how accurately isolated cells reflect responses of intact organs. In this work, we compare transcriptional responses in livers of Sprague-Dawley rats and primary hepatocyte cells after exposure to RDX to determine how faithfully the in vitro model system reflects in vivo responses.ResultsExpression patterns were found to be markedly different between liver tissue and primary cell cultures before exposure to RDX. Liver gene expression was enriched in processes important in toxicology such as metabolism of amino acids, lipids, aromatic compounds, and drugs when compared to cells. Transcriptional responses in cells exposed to 7.5, 15, or 30 mg/L RDX for 24 and 48 hours were different from those of livers isolated from rats 24 hours after exposure to 12, 24, or 48 mg/Kg RDX. Most of the differentially expressed genes identified across conditions and treatments could be attributed to differences between cells and tissue. Some similarity was observed in RDX effects on gene expression between tissue and cells, but also significant differences that appear to reflect the state of the cell or tissue examined.ConclusionLiver tissue and primary cells express different suites of genes that suggest they have fundamental differences in their cell physiology. Expression effects related to RDX exposure in cells reflected a fraction of liver responses indicating that care must be taken in extrapolating from primary cells to whole animal organ toxicity effects.

The determination of tungsten, molybdenum, and phosphorus oxyanions by high performance liquid chromatography inductively coupled plasma mass spectrometery.

The toxic properties of tungsten compounds have recently been brought to the forefront with clusters of human cancer cases, such as in Fallon, NV. Such instances have made the determination of tungsten in natural water supplies vitally important. Tungsten exists in most environmental matrices as the soluble and mobile tungstate anion, although it can polymerize with itself and other anions, such as molybdate and phosphate. Because the geochemical and toxicological properties of these polymer species will vary from the monomeric tungstate parent, determination of tungstate speciation is as critical as determination of total dissolved tungsten concentration. Use of chromatographic separations, followed by element-specific detection is a proven technology for elemental speciation. In the present work, anion exchange chromatography has been coupled to inductively coupled plasma mass spectrometry to determine tungstate, molybdate, and phosphate species at the sub-microg l(-1) and microg l(-1) levels. The method provides quantitative determination of these species in about 10 min with the capability to simultaneously determine other oxyanion species. The method has been applied to groundwater and extracts of soils amended with tungsten powder. The water soluble tungsten in 1-h deionized water extracts after six months of soil aging was >15 mg l(-1), however, only approximately 50% of the tungsten was present as monomeric tungstate.

Comparison of macro-gravimetric and micro-colorimetric lipid determination methods.

In order to validate a method for lipid analysis of small tissue samples, the standard macro-gravimetric method of Bligh-Dyer (1959) [E.G. Bligh, W.J. Dyer, Can. J. Biochem. Physiol. 37 (1959) 911] and a modification of the micro-colorimetric assay developed by Van Handel (1985) [E. Van Handel, J. Am. Mosq. Control Assoc. 1 (1985) 302] were compared. No significant differences were observed for wet tissues of two species of fish. However, limited analysis of wet tissue of the amphipod, Leptocheirusplumulosus, indicated that the Bligh-Dyer gravimetric method generated higher lipid values, most likely due to the inclusion of non-lipid materials. Additionally, significant differences between the methods were observed with dry tissues, with the micro-colorimetric method consistently reporting calculated lipid values greater than as reported by the gravimetric method. This was most likely due to poor extraction of dry tissue in the standard Bligh-Dyer method, as no significant differences were found when analyzing a single composite extract. The data presented supports the conclusion that the micro-colorimetric method described in this paper is accurate, rapid, and minimizes time and solvent use.

Tungsten effects on survival, growth, and reproduction in the earthworm, Eisenia fetida.

To provide basic toxicity data for formulating risk characterization benchmarks, the effects of tungsten on survival, growth, and reproduction were investigated in the earthworm Eisenia fetida. Parallel studies with lead as a reference toxicant also were conducted. Although sodium tungstate (Na2WO4) was less acutely toxic than lead nitrate (Pb(NO3)2) in 14-d spiked field soil acute toxicity assays (lethal concentrations for 50% of organisms: W, 6,250 mg/kg; Pb, 2,490 mg/kg), tungstate completely inhibited reproduction in 28- and 56-d assays at all tested tungsten concentrations (> or = 704 mg/kg). By comparison, cocoon production was not significantly reduced for lead concentrations until concentrations reached 766 mg/kg, and cocoon production was still observed at the highest concentration tested (1,650 mg/kg). These data indicate that tungsten is a reproductive toxicant for earthworms and that, by comparison, its sublethal toxicity is greater than that of lead. Toxicity data for other soil invertebrate species are required to fully establish benchmark levels/ecological soil screening levels for tungsten.

Tungsten Effects on Phosphate-Dependent Biochemical Pathways are Species- and Liver Cell Line-Dependent

Tungsten, in the form of tungstate, polymerizes with phosphate, and as extensive polymerization occurs, cellular phosphorylation and dephosphorylation reactions may be disrupted, resulting in negative effects on cellular functions. A series of studies were conducted to evaluate the effect of tungsten on several phosphate-dependent intracellular functions, including energy cycling (ATP), regulation of enzyme activity (cytosolic protein tyrosine kinase [cytPTK] and tyrosine phosphatase), and intracellular secondary messengers (cyclic adenosine monophosphate [cAMP]). Rat noncancerous hepatocyte (Clone-9), rat cancerous hepatocyte (H4IIE), and human cancerous hepatocyte (HepG2) cells were exposed to 1-1000 mg/l tungsten (in the form of sodium tungstate) for 24 h, lysed, and analyzed for the above biochemical parameters. Cellular ATP levels were not significantly affected in any cell line. After 4 h, tungsten significantly decreased cytPTK activity in Clone-9 cells at >or= 18 mg/l, had no effect in H4IIE cells, and significantly increased cytPTK activity by 70% in HepG2 cells at >or= 2 mg/l. CytPTK displayed a slight hormetic response to tungsten after 24-h exposure yet returned to normal after 48-h exposure. Tungsten significantly increased cAMP by over 60% in Clone-9 cells at >or= 100 mg/l, significantly increased cAMP in H4IIE cells at only 100 mg/l, and significantly increased cAMP in HepG2 cells between 1-100 mg/l but at much more modest levels (8-20%). In conclusion, these data indicate that tungsten produces complex results that must be carefully interpreted in the context of their respective animal models, as well as the phenotype of the cell lines (i.e., normal vs. cancerous).

PROFILING THE DEPTH AND EXTENT OF CONTAMINATION OF A SHIP CHANNEL USING THE P450RGS CELL-BASED ASSAY FOR DIOXINS AND RELATED COMPOUNDS

A rapid and sensitive cell-based screening assay for dioxin equivalences (TCDD EQs) in dredged sediments has been developed. The P450RGS assay employs a transgenic human hepatoma cell line in which the firefly luciferase gene, Zuc, has been stably inserted as a reporter. P450RGS conforms to APHA Standard Method 8070 and ASTM Standard E-1853. EPA has promulgated the assay as EPA Method 4425 in update IVA of the EPA SW846 Methods Manual. Modifications to the method made at USACE ERDC-WES include a combined extraction/cleanup procedure using accelerated solvent extraction (ASE) with a sulfuric acid/silica gel packing in the extraction cell, and a 96-well microtiter plate format for the assay. These modifications substantially increase the volume of sample throughput and reduce the cost of the assay. Assays in which TCDD EQs are reported are typically less than l/lOth the cost of dioxin analysis using GC/MS, and have comparable sensitivity. In this study 13 stations were designated at intervals extending from the mouth to the apex of a navigation channel requiring dredging. Cores from 3 to 12-l? sediment depth were sectioned and assayed for TCDD EQs using P450RGS. Seven of the cores were also analyzed by conventional chemical methods for chemical contaminants. Additional cores were taken for bioaccumulation and toxicity testing. The pattern and relative magnitudes of contamination found with the chemical analyses was highly correlated with that identified by the P450RGS assay (r2 = 0.816 to 0.892, P << 0.001). Additionally, the P450RGS results provided a surrogate for highly expensive GC/MS analysis for dioxins at a small fraction of the cost of chemical analysis. More extensive sampling than would have been affordable using only conventional chemistry resulted in a fuller characterization of the extent and depth of contamination, and demonstrated the utility of the cell-based assay as a screening and monitoring tool for navigation channel sediments.

Comparison of Gene Expression Effects in Liver Tissue and Primary Hepatocyte Cell Cultures After Exposure to Hexahydro-1, 3, 5Trinitro-1, 3, 5-Triazine

We examined Sprague-Dawley rat liver tissue and primary hepatocyte cells to understand how they compared in gene expression responses to hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Gene expression was analyzed in primary cell cultures exposed to 7.5, 15, or 30 mg/L RDX for 24 and 48 hours. Cells were compared to liver from rats 24 hours after exposure to 12, 24, or 48 mg/Kg RDX. Similarity of present/absent calls to liver in control exposures was 88% at 24 hrs and 79% at 48 hrs. Significant changes were identified using mixed-model analysis of variance. Up to 6.4% of differentially expressed genes present in liver were differentially expressed in 24 hr cells and less than 5.5% in 48 hr cells. Of genes with identifiable KEGG pathways, 24 hr cells 78.4% similar to liver pathways and 48 hr cells were 44.3% similar. These data suggest care should be taken extrapolating effects from in vitro to in vivo levels