Zebrafish Models for Human Acute Organophosphorus Poisoning.

Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

Differential transcription of fathead minnow immune-related genes following infection with frog virus 3, an emerging pathogen of ectothermic vertebrates.

Frog virus 3 (FV3) and other ranaviruses are responsible for die-offs involving wild, farmed, and captive amphibians, fish, and reptiles. To ascertain which elements of the immune system respond to infection, we explored transcriptional responses following infection of fathead minnow cells with either wild type (wt) FV3 or a knock out (KO) mutant targeting the 18 kDa immediate early gene (18K). At 8h post infection we observed marked upregulation of multiple transcripts encoding proteins affecting innate and acquired immunity. Sequences expressed 4-fold or higher in wt-infected cells included transcripts encoding interferon (IFN), IFN regulatory factors (IRFs), IFN stimulated genes (ISGs) such as Mx and MHC class I, and interleukins IL-1ß, IL-8, IL-17C and IL-12. Cells infected with the 18K KO mutant (∆18K) showed qualitative differences and lower levels of induction. Collectively, these results indicate that ranavirus infection induced expression of multiple cellular genes affecting both innate and acquired immunity.

Differential effects and potential adverse outcomes of ionic silver and silver nanoparticles in vivo and in vitro.

Nanoparticles are of concern because of widespread use, but it is unclear if metal nanoparticles cause effects directly or indirectly. We explored whether polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) cause effects through intact nanoparticles or dissolved silver. Females of the model species fathead minnow (Pimephales promelas) were exposed to either 4.8 µg/L of AgNO3 or 61.4 µg/L of PVP-AgNPs for 96h. Microarray analyses were used to identify impacted receptors and toxicity pathways in liver and brain tissues that were confirmed using in vitro mammalian assays. AgNO3 and PVP-AgNP exposed fish had common and distinct effects consistent with both intact nanoparticles and dissolved silver causing effects. PVP-AgNPs and AgNO3 both affected pathways involved in Na(+), K(+), and H(+) homeostasis and oxidative stress but different neurotoxicity pathways. In vivo effects were supported by PVP-AgNP activation of five in vitro nuclear receptor assays and inhibition of ligand binding to the dopamine receptor. AgNO3 inhibited ligand binding to adrenergic receptors α1 and α2 and cannabinoid receptor CB1, but had no effect in nuclear receptor assays. PVP-AgNPs have the potential to cause effects both through intact nanoparticles and metal ions, each interacting with different initiating events. Since the in vitro and in vivo assays examined here are commonly used in human and ecological hazard screening, this work suggests that environmental health assessments should consider effects of intact nanoparticles in addition to dissolved metals.

Effects of BDE-209 contaminated sediments on zebrafish development and potential implications to human health.

Polybrominated diphenyl ethers are compounds widely used as flame-retardants, which are of increasing environmental concern due to their persistence, and potential adverse effects. This study had two objectives. First, we assessed if BDE-209 in sediment was bioavailable and bioaccumulated into zebrafish embryos. Secondly, we assessed the potential impact on human and environmental health of bioavailable BDE-209 using human in vitro cell assays and zebrafish embryos. Zebrafish were exposed from 4h to 8days post-fertilization to sediments spiked with 12.5mg/kg of BDE-209. Zebrafish larvae accumulated ten fold more BDE-209 than controls in unspiked sediment after 8days. BDE-209 impacted expression of neurological pathways and altered behavior of larvae, although BDE-209 had no visible affect on thyroid function or motoneuron and neuromast development. Zebrafish data and in silico predictions suggested that BDE-209 would also interact with key human transcription factors and receptors. We therefore tested these predictions using mammalian in vitro assays. BDE-209 activated human aryl hydrocarbon receptor, peroxisome proliferator activating receptors, CF/b-cat, activator protein 1, Oct-MLP, and the estrogen receptor-related alpha (ERRα) receptor in cell-based assays. BDE-209 also inhibited human acetylcholinesterase activity. The observation that BDE-209 can be bioaccumulated from contaminated sediment highlights the need to consider this as a potential environmental exposure route. Once accumulated, our data also show that BDE-209 has the potential to cause impacts on both human and environmental health.

The good, the bad, and the toxic: approaching hormesis in Daphnia magna exposed to an energetic compound.

A hormetic response is characterized by an opposite effect in small and large doses of chemical exposure, often resulting in seemingly beneficial effects at low doses. Here, we examined the potential mechanisms underlying the hormetic response of Daphnia magna to the energetic trinitrotoluene (TNT). Daphnia magna were exposed to TNT for 21 days, and a significant increase in adult length and number of neonates was identified at low concentrations (0.002-0.22 mg/L TNT), while toxic effects were identified at high concentrations (0.97 mg/L TNT and above). Microarray analysis of D. magna exposed to 0.004, 0.12, and 1.85 mg/L TNT identified effects on lipid metabolism as a potential mechanism underlying hormetic effects. Lipidomic analysis of exposed D. magna supported the hypothesis that TNT exposure affected lipid and fatty acid metabolism, showing that hormetic effects could be related to changes in polyunsaturated fatty acids known to be involved in Daphnia growth and reproduction. Our results show that Daphnia exposed to low levels of TNT presented hormetic growth and reproduction enhancement, while higher TNT concentrations had an opposite effect. Our results also show how a systems approach can help elucidate potential mechanisms of action and adverse outcomes.

Transcriptional signature of progesterone in the fathead minnow ovary (Pimephales promelas).

A growing number of studies have examined transcriptional responses to sex steroids along the hypothalamic-pituitary-gonadal axis in teleost fishes. However, data are lacking on the molecular cascades that underlie progesterone signaling. The objective of this study was to characterize the transcriptional response in the ovary of fathead minnows (Pimephales promelas) in response to progesterone (P4). Fathead minnow ovaries were exposed in vitro to 500 ng P4/L. Germinal vesicle migration and breakdown (GVBD) was observed and microarrays were used to identify gene cascades affected by P4. Microarray analysis identified 1702 differentially expressed transcripts after P4 treatment. Functional enrichment analysis revealed that transcripts involved in the molecular functions of protein serine/threonine kinase activity, ATP binding, and activity of calcium channels were increased after P4 treatment. There was an overwhelming decrease in levels of transcripts of genes that are structural constituents of ribosomes with P4 treatment. There was also evidence for gene expression changes in steroid and maturation-related transcripts. Pathway analyses identified cell cycle regulation, insulin action, hedgehog, and B cell activation as pathways containing an over-representation of highly regulated transcripts. Significant regulatory sub-networks of P4-mediated transcripts included genes regulated by tumor protein p53 and E2F transcription factor 1. These data provide novel insight into the molecular signaling cascades that underlie P4-signaling in the ovary and identify genes and processes that may indicate premature GVBD due to environmental pollutants that mimic progestins.

Building quantitative prediction models for tissue residue of two explosives compounds in earthworms from microarray gene expression data.

Soil contamination near munitions plants and testing grounds is a serious environmental concern that can result in the formation of tissue chemical residue in exposed animals. Quantitative prediction of tissue residue still represents a challenging task despite long-term interest and pursuit, as tissue residue formation is the result of many dynamic processes including uptake, transformation, and assimilation. The availability of high-dimensional microarray gene expression data presents a new opportunity for computational predictive modeling of tissue residue from changes in expression profile. Here we analyzed a 240-sample data set with measurements of transcriptomic-wide gene expression and tissue residue of two chemicals, 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), in the earthworm Eisenia fetida. We applied two different computational approaches, LASSO (Least Absolute Shrinkage and Selection Operator) and RF (Random Forest), to identify predictor genes and built predictive models. Each approach was tested alone and in combination with a prior variable selection procedure that involved the Wilcoxon rank-sum test and HOPACH (Hierarchical Ordered Partitioning And Collapsing Hybrid). Model evaluation results suggest that LASSO was the best performer of minimum complexity on the TNT data set, whereas the combined Wilcoxon-HOPACH-RF approach achieved the highest prediction accuracy on the RDX data set. Our models separately identified two small sets of ca. 30 predictor genes for RDX and TNT. We have demonstrated that both LASSO and RF are powerful tools for quantitative prediction of tissue residue. They also leave more unknown than explained, however, allowing room for improvement with other computational methods and extension to mixture contamination scenarios.

Ecotoxicogenomics to support ecological risk assessment: a case study with bisphenol A in fish.

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.

Assessment of chemical mixtures and groundwater effects on Daphnia magna transcriptomics.

Small organisms can be used as biomonitoring tools to assess chemicals in the environment. Chemical stressors are especially hard to assess and monitor when present as complex mixtures. Here, fifteen polymerase chain reaction assays targeting Daphnia magna genes were calibrated to responses elicited in D. magna exposed for 24 h to five different doses each of the munitions constituents 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, trinitrobenzene, dinitrobenzene, or 1,3,5-trinitro-1,3,5-triazacyclohexane. A piecewise-linear model for log-fold expression changes in gene assays was used to predict response to munitions mixtures and contaminated groundwater under the assumption that chemical effects were additive. The correlations of model predictions with actual expression changes ranged from 0.12 to 0.78 with an average of 0.5. To better understand possible mixture effects, gene expression changes from all treatments were compared using high-density microarrays. Whereas mixtures and groundwater exposures had genes and gene functions in common with single chemical exposures, unique functions were also affected, which was consistent with the nonadditivity of chemical effects in these mixtures. These results suggest that, while gene behavior in response to chemical exposure can be partially predicted based on chemical exposure, estimation of the composition of mixtures from chemical responses is difficult without further understanding of gene behavior in mixtures. Future work will need to examine additive and nonadditive mixture effects using a much greater range of different chemical classes in order to clarify the behavior and predictability of complex mixtures.

Tracheal occlusion conditioning in conscious rats modulates gene expression profile of medial thalamus.

The thalamus may be the critical brain area involved in sensory gating and the relay of respiratory mechanical information to the cerebral cortex for the conscious awareness of breathing. We hypothesized that respiratory mechanical stimuli in the form of tracheal occlusions would modulate the gene expression profile of the thalamus. Specifically, it was reasoned that conditioning to the respiratory loading would induce a state change in the medial thalamus consistent with a change in sensory gating and the activation of molecular pathways associated with learning and memory. In addition, respiratory loading is stressful and thus should elicit changes in gene expressions related to stress, anxiety, and depression. Rats were instrumented with inflatable tracheal cuffs. Following surgical recovery, they underwent 10 days (5 days/week) of transient tracheal occlusion conditioning. On day 10, the animals were sacrificed and the brains removed. The medial thalamus was dissected and microarray analysis of gene expression performed. Tracheal obstruction conditioning modulated a total of 661 genes (p < 0.05, log(2) fold change ≥0.58), 250 genes were down-regulated and 411 up-regulated. There was a significant down-regulation of GAD1, GAD2 and HTR1A, HTR2A genes. CCK, PRKCG, mGluR4, and KCJN9 genes were significantly up-regulated. Some of these genes have been associated with anxiety and depression, while others have been shown to play a role in switching between tonic and burst firing modes in the thalamus and thus may be involved in gating of the respiratory stimuli. Furthermore, gene ontology and pathway analysis showed a significant modulation of learning and memory pathways. These results support the hypothesis that the medial thalamus is involved in the respiratory sensory neural pathway due to the state change of its gene expression profile following repeated tracheal occlusions.

Behavioral and genomic impacts of a wastewater effluent on the fathead minnow.

Rivers containing effluents from water treatment plants are complex soups of compounds, ranging from pharmaceuticals to natural hormones. Male fathead minnows (Pimephales promelas) were exposed for 3 weeks to effluent waters from the Metropolitan Wastewater Treatment Plant in St. Paul, MN. Fish were tested for their competitive nest holding behavior. Changes in vitellogenin were measured and these were correlated to changes in gene expression using a 22,000 gene microarray developed specifically for fathead minnows. Significant changes in gene expression were observed in both liver and testis, which correlate to phenotypic changes of vitellogenin induction and reduced competitive behavior. We also compared by real-time PCR the expression changes in key genes related to steroid biosynthesis and metabolism in fish exposed to the effluent as well as in fish exposed to a model estrogen and a model androgen. While the gene expression signature from effluent-exposed fish shared some elements with estrogen and androgen signatures, overall it was different, underscoring the complexity of compounds present in sewage and their different modes of action.

Biomarker discovery and transcriptomic responses in Daphnia magna exposed to munitions constituents.

Ecotoxicogenomic approaches are emerging as alternative methods in environmental monitoring because they allow insight into pollutant modes of action and help assess the causal agents and potential toxicity beyond the traditional end points of death, growth, and reproduction. Gene expression analysis has shown particular promise for identifying gene expression biomarkers of chemical exposure that can be further used to monitor specific chemical exposures in the environment. We focused on the development of gene expression markers to detect and discriminate between chemical exposures. Using a custom cDNA microarray for Daphnia magna, we identified distinct expression fingerprints in response to exposure at sublethal concentrations of Cu, Zn, Pb, and munitions constituents. Using the results obtained from microarray analysis, we chose a suite of potential biomarkers for each of the specific exposures. The selected potential biomarkers were tested in independent chemical exposures for specificity using quantitative reverse transcription polymerase chain reaction. Six genes were confirmed as differentially regulated bythe selected chemical exposures. Furthermore, each exposure was identified by response of a unique combination (suite) of individual gene expression biomarkers. These results demonstrate the potential for discovery and validation of novel biomarkers of chemical exposures using gene expression analysis, which could have broad applicability in environmental monitoring.

Dissolution, sorption, and kinetics involved in systems containing explosives, water, and soil.

Knowledge of explosives sorption and transformation processes is required to ensure that the proper fate and transport of such contaminants is understood at military ranges and ammunition production sites. Bioremediation of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and related nitroaromatic compounds has met with mixed success, which is potentially due to the uncertainty of how energetic compounds are bound to different soil types. This study investigated the dissolution and sorption properties of TNT and RDX explosives associated with six different soil types. Understanding the associations that explosives have with a different soil type assists with the development of conceptual models used for the sequestration process, risk analysis guidelines, and site assessment tools. In three-way systems of crystalline explosives, soil, and water, the maximum explosive solubility was not achieved due to the sorption of the explosive onto the soil particles and observed production of transformation byproducts. Significantly different sorption effects were also observed between sterile (gamma-irradiated) and nonsterile (nonirradiated) soils with the introduction of crystalline TNT and RDX into soil-water systems.

Uptake of hexanitrohexaazaisowurtzitane (CL-20) by the earthworm Eisenia fetida through dermal contact.

The explosive compound hexanitrohexaazaisowurtzitane (CL-20) has been shown to cause both lethal and sublethal (reproductive and neurotoxic) effects in exposed oligochaetes. However, whether worms take up CL-20 and how much CL-20 enters worm bodies leading to toxicity (e.g., lethality) remain to be determined. In the present study, we used high performance liquid chromatography (HPLC) and radiolabeled tracer methods to investigate the CL-20 uptake in the whole worm body after contact exposures. Worms (Eisenia fetida) were exposed to filter paper spiked with non-radioactive or [U-(14)C]-labeled CL-20 for 1-3 d. The radiolabeled tracer method allowed us to detect the parent compound and transformation products in worms exposed to as low as 0.04 microg CL-20 cm(-2) of filter paper. The HPLC method without radiolabeled tracer was far less sensitive with a detection limit of 2.17 microg CL-20 cm(-2). Using the radiolabeled tracer, we were able to demonstrate that the worm body concentration linearly correlated to the filter paper concentration < or =0.34 microg cm(-2) (r=0.94) if no breakdown products are assumed. At higher concentrations, the body concentration increased slowly and saturated at around 11 microg g(-1) dry mass resulting in an estimated lethal critical body burden of 10-15 microg CL-20 g(-1) dry mass. These findings demonstrate that CL-20 or potential transformation products are taken into the earthworm body through dermal contact. This information should prove valuable in assessing the bioaccumulation potential and ecological risks of CL-20.

Subacute toxicity of oral 2,6-dinitrotoluene and 1,3,5-trinitro-1,3,5-triazine (RDX) exposure to the northern bobwhite (Colinus virginianus).

Military activities associated with training have resulted in soil residues of munition compounds and their breakdown products. Often, these areas are valuable habitats used by birds. To evaluate the possibility of adverse effects in birds, the effects from oral exposures of 2,6-dinitrotoluene (2,6-DNT) and 1,3,5-trinitro-1,3,5-triazine (RDX) were tested using a controlled dosing regime in northern bobwhite (Colinus virginianus). Nine groups of birds of mixed sex received either corn oil or 50, 100, 190, or 350 mg 2,6-DNT/kg body weight/d or 20, 80, 125, or 180 mg RDX/kg body weight/d mixed in corn oil via gavage for 14 d. Etiology of disease was markedly different between compounds. Increased RDX exposure caused an inverse relationship to time of death, symptoms including clonic followed by tonic convulsions, and death shortly thereafter. Brain concentrations of RDX postmortem, however, were relatively consistent (mean +/- standard error, 20.5 +/- 2.9 mg/kg tissue). Observations of 2,6-DNT effects included gastrointestinal distress, dehydration, and a reduction in body mass and feed consumption. Together, these data suggest that RDX is more toxic from short, repeated exposures than 2,6-DNT, resulting in central nervous system-related effects, whereas 2,6-DNT causes gastrointestinal disturbances at higher exposures.