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).

Subcellular compartmentalization of lead in the earthworm, Eisenia fetida: Relationship to survival and reproduction.

Metals are detoxified and sequestered into subcellular compartments when accumulated by earthworms. Differential centrifugation was used to quantify subcellular Pb in three separate studies to measure 14-day acute toxicity (lethality), 28/56-day reproductive effects, and 90-day bioaccumulation in spiked-soil exposed earthworms, Eisenia fetida. Observed toxicity and total body Pb was consistent with published work of others. Pb showed concentration-dependent toxicity relationships (lethality and reproduction) for total and subcellular Pb. Toxic fraction and total Pb showed similar concentration-response patterns in the 14-day and 28/56-day studies and tended to increase towards a plateau at higher concentrations. Linear correlations of subcellular to total Pb was observed in all studies except the 90-day bioaccumulation study in which toxic fraction Pb appeared to approach a maximum over the period between Day 56 and Day 90. In a follow-on study using two different contaminated soil types, toxic fraction and total Pb concentrations as related to reproductive effects were consistent with data from our spiked soil studies, and this suggests it may be possible to use such values to "factor out" matrix-specific influences that otherwise skew toxicity values when expressed relative to soil concentrations. Our findings, however, suggest the subcellular fractionation approach may not offer advantages over total Pb determination in short-term exposure studies but may become important when longer exposure periods (greater than 90 days) are considered. In this respect, the technique we describe has the potential to provide valuable information for assessing and interpreting Pb toxicity as a function of earthworm body burden.

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

BACKGROUND: Explosive 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. RESULTS: Sublethal 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. CONCLUSIONS: Sublethal 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.

Cloning, analysis and functional annotation of expressed sequence tags from the Earthworm Eisenia fetida.

BACKGROUND: Eisenia 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. RESULTS: A 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. CONCLUSION: The ESTMD containing the sequence and annotation information of 4032 E. fetida ESTs is publicly accessible at http://mcbc.usm.edu/estmd/.

Comparative neurotoxicity of two energetic compounds, hexanitrohexaazaisowurtzitane and hexahydro-1,3,5-trinitro-1,3,5-triazine, in the earthworm Eisenia fetida.

Hexanitrohexaazaisowurtzitane (CL-20) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), both energetic compounds, share some degree of structural similarity. A noninvasive electrophysiological technique was employed to assess the impacts of acute sublethal exposures on impulse conduction in medial (MGF) and lateral (LGF) giant nerve fiber pathways of the earthworm Eisenia fetida and to evaluate the reversibility of neurotoxic effects. Earthworms were exposed to either 0.02 to 2.15 microg/cm2 of CL-20 or 0.04 to 5.35 microg/cm2 of RDX, for 1 to 14 d, on moistened filter paper. Conduction velocities of MGF and LGF were recorded on a digital oscilloscope before and after exposure. Results indicate that at exposure levels as low as 0.02 microg/cm2 of CL-20 or 0.21 microg/cm2 of RDX, worms exhibited physiological impacts such as retardation, stiffness, and body shrink. Both MGF and LGF conduction velocities were negatively correlated with increasing doses of CL-20 or RDX. However, such neurotoxic effects were alleviated or even eliminated within a few days after exposed worms were transferred to an uncontaminated environment, indicating that the neurotoxicity is reversible even after 6-d exposure. The CL-20 is more potent than RDX, which is consistent with previous studies on lethality, growth, and reproduction endpoints in soil oligochaetes.

Assessment of lead uptake in reptilian prey species.

As part of an investigation determining the trophically available fraction of metals in a model terrestrial food web, i.e., invertebrate prey to Western fence lizards (Sceloporus occidentalis), we evaluated the ability of several invertebrate prey to bioaccumulate lead and to form metals-rich granules, which are hypothesized to be non-available to predators. Crickets (Acheta domestica), tenebroid beetle larvae (Tenebrio molitor), and isopods (Porcellio scaber) were selected as model prey organisms. Lack of standard exposure methodologies for these species has presented a barrier to trophic transfer evaluations, as each species has particular requirements that create challenges for designing exposure conditions. We were able to devise exposure conditions for all three species that allow long-term exposure studies. All prey organisms accumulated lead from contaminated food, and for all species the majority of the accumulated Pb was associated with the exoskeleton (>50%), with metals-rich granules accounting for most of the remaining accumulated lead.

Toxicogenomic analysis provides new insights into molecular mechanisms of the sublethal toxicity of 2,4,6-trinitrotoluene in Eisenia fetida.

Xenobiotics such as explosives and pesticides released into the environment can have lethal and sublethal impacts on soil organisms such as earthworms with potential subsequent impacts at highertrophic levels. To better understand the molecular toxicological mechanisms of 2,4,6-trinitrotoluene (TNT), a commonly used explosive, in Eisenia fetida, earthworms were exposed to a gradient of TNT-spiked soils for 28 days and impacts on gene expression were examined using a 4032 cDNA microarray. Reproduction was increased at low doses of TNT, whereas high doses of TNT reduced juvenile production. On the basis of reproduction responses to TNT, four treatments, that is, control, 2, 10.6, and 38.7 mg/kg, were selected for gene expression studies in a balanced interwoven loop design microarray experiment in which the expression of 311 transcripts was significantly affected. Reverse-transcription quantitative polymerase chain reaction (RT-QPCR) data on 68 selected differentially and nondifferentially expressed transcripts showed a significant correlation with microarray results. The expression of genes involved in multiple biological processes was altered, including muscle contraction, neuronal signaling and growth, ubiquitinylation, fibrinolysis and coagulation, iron and calcium homeostasis, oxygen transport, and immunity. Chitinase activity assays confirmed down-regulation of chitinase genes as indicated by array and RT-QPCR data. An acute toxicity test provided evidence that dermal contact with TNT can cause bleeding, inflammation, and constriction, which may be explained by gene expression results. Sublethal doses of TNT affected the nervous system, caused blood disorders similar to methemoglobinemia, and weakened immunity in E. fetida.

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.

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

BACKGROUND: Cell 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. RESULTS: Expression 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. CONCLUSION: Liver 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.

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.

Up-and-down procedure (UDP) determinations of acute oral toxicity of nitroso degradation products of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a widely used military explosive and soil and ground water contaminant of munitions manufacturing and artillery training sites, undergoes microbial nitroreductase metabolism to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Human occupational and accidental exposures to RDX, as well as acute oral exposures in rats, result in seizures, but little is known about the toxicity of the RDX degradation products. The main objective of the present study was to determine the oral LD50 of the most potent RDX N-nitroso product in female Sprague-Dawley rats using the recently validated up-and-down procedure (UDP). With only 26 rats, MNX was identified as the most potent metabolite and a maximum likelihood estimate of 187 mg kg(-1) (95% confidence interval 118-491 mg kg(-1)) for its LD50 was established and found equivalent to that of RDX determined with the same protocol. CNS toxicity, manifested as forelimb clonic seizures progressing to generalized clonic-tonic seizures, was the critical adverse effect. Further, confirmation of the UDP LD50 for MNX with a fixed-dose design enabled identification of 94 mg kg(-1) as the highest nonlethal dose. An ED50 of 57 mg kg(-1) was determined for neurotoxicity, while splenic hemosiderosis and decreased blood hematocrit and hemoglobin concentration occurred with a threshold at 94 mg kg(-1) in 14-day survivors. These studies, while providing new toxicity data necessary for the management of RDX-contaminated sites, illustrate the efficiency of the UDP for comparative acute toxicity determinations and its value in guiding further characterization of dose dependency of identified adverse effects.