Mitchell S. Wilbanks

Analysis of Common and Specific Mechanisms of Liver Function Affected by Nitrotoluene Compounds

Background Nitrotoluenes are widely used chemical manufacturing and munitions applications. This group of chemicals has been shown to cause a range of effects from anemia and hypercholesterolemia to testicular atrophy. We have examined the molecular and functional effects of five different, but structurally related, nitrotoluenes on using an integrative systems biology approach to gain insight into common and disparate mechanisms underlying effects caused by these chemicals. Methodology/Principal Findings Sprague-Dawley female rats were exposed via gavage to one of five concentrations of one of five nitrotoluenes [2,4,6-trinitrotoluene (TNT), 2-amino-4,6-dinitrotoluene (2ADNT) 4-amino-2,6-dinitrotoulene (4ADNT), 2,4-dinitrotoluene (2,4DNT) and 2,6-dinitrotoluene (2,6DNT)] with necropsy and tissue collection at 24 or 48 h. Gene expression profile results correlated well with clinical data and liver histopathology that lead to the concept that hematotoxicity was followed by hepatotoxicity. Overall, 2,4DNT, 2,6DNT and TNT had stronger effects than 2ADNT and 4ADNT. Common functional terms, gene expression patterns, pathways and networks were regulated across all nitrotoluenes. These pathways included NRF2-mediated oxidative stress response, aryl hydrocarbon receptor signaling, LPS/IL-1 mediated inhibition of RXR function, xenobiotic metabolism signaling and metabolism of xenobiotics by cytochrome P450. One biological process common to all compounds, lipid metabolism, was found to be impacted both at the transcriptional and lipid production level. Conclusions/Significance A systems biology strategy was used to identify biochemical pathways affected by five nitroaromatic compounds and to integrate data that tie biochemical alterations to pathological changes. An integrative graphical network model was constructed by combining genomic, gene pathway, lipidomic, and physiological endpoint results to better understand mechanisms of liver toxicity and physiological endpoints affected by these compounds.

Conserved toxic responses across divergent phylogenetic lineages: a meta-analysis of the neurotoxic effects of RDX among multiple species using toxicogenomics

At military training sites, a variety of pollutants such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), may contaminate the area originating from used munitions. Studies investigating the mechanism of toxicity of RDX have shown that it affects the central nervous system causing seizures in humans and animals. Environmental pollutants such as RDX have the potential to affect many different species, therefore it is important to establish how phylogenetically distant species may respond to these types of emerging pollutants. In this paper, we have used a transcriptional network approach to compare and contrast the neurotoxic effects of RDX among five phylogenetically disparate species: rat (Sprague-Dawley), Northern bobwhite quail (Colinus virginianus), fathead minnow (Pimephales promelas), earthworm (Eisenia fetida), and coral (Acropora formosa). Pathway enrichment analysis indicated a conservation of RDX impacts on pathways related to neuronal function in rat, Northern bobwhite quail, fathead minnows and earthworm, but not in coral. As evolutionary distance increased common responses decreased with impacts on energy and metabolism dominating effects in coral. A neurotransmission related transcriptional network based on whole rat brain responses to RDX exposure was used to identify functionally related modules of genes, components of which were conserved across species depending upon evolutionary distance. Overall, the meta-analysis using genomic data of the effects of RDX on several species suggested a common and conserved mode of action of the chemical throughout phylogenetically remote organisms.

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.

Comparative cytotoxicity of alachlor, acetochlor, and metolachlor herbicides in isolated rat and cryopreserved human hepatocytes.

Noncancerous adverse effects observed at the lowest dose for chloroacetanilide herbicides alachlor [2‐chloro‐2′,6′‐diethyl‐N‐(methoxymethyl)‐acetanilide] and acetochlor [2‐chloro‐2′‐methyl‐6′‐ethyl‐N‐(ethoxymethyl)acetanilide], but not metolachlor [2‐chloro‐2′‐ethyl‐6′‐methyl‐N‐(1‐methyl‐2‐methoxymethyl)acetanilide], are hepatotoxicity in rats and dogs. Liver microsomal N‐dealkylation, a step in the putative activating pathway, of acetochlor exceeds that of alachlor and is negligible for metolachlor. In the present investigation, cytotoxicity of the three chloroacetanilides was ranked using isolated rat and cryopreserved human hepatocytes to correlate this endpoint with CYP3A‐dependent metabolism. Chloroacetanilide cytotoxicity in rat hepatocyte suspensions was time dependent (e.g., LC50 ‐ alachlor/2 h vs. LC50 ‐ alachlor/4 h = 765 vs. 325 μM). Alachlor and acetochlor were more potent than metolachlor after 2 and 4 h, times when N‐dealkylated alachlor product 2‐chloro‐N‐(2,6‐diethylphenyl)acetamide (CDEPA) formation was readily detectable. Alachlor and acetochlor potencies with cryopreserved human hepatocytes at 2 h were comparable to freshly isolated rat hepatocytes, and alachlor metabolism to CDEPA was likewise detectable. Unlike rat hepatocytes, metolachlor potency was equivalent to acetochlor and alachlor in human hepatocytes. Furthermore, chloroacetanilide cytotoxicity from two sources of human hepatocytes varied inversely with CYP3A4 activity. Collectively, while cytotoxicity in rat hepatocytes was consistent with chloroacetanilide activation by CYP3A, an activating role for CYP3A4 was not supported with human hepatocytes.

A weight of evidence assessment approach for adverse outcome pathways.

The adverse outcome pathway (AOP) is a framework to mechanistically link molecular initiating events to adverse biological outcomes. From a regulatory perspective, it is of crucial importance to determine the confidence for the AOP in question as well as the quality of data available in supporting this evaluation. A weight of evidence approach has been proposed for this task, but many of the existing frameworks for weight of evidence evaluation are qualitative and there is not clear guidance regarding how weight of evidence should be calculated for an AOP. In this paper we advocate the use of a subject matter expertise driven approach for weight of evidence evaluation based on criteria and metrics related to data quality and the strength of causal linkages between key events. As a demonstration, we notionally determine weight of evidence scores for two AOPs: Non-competitive ionotropic GABA receptor antagonism leading to epileptic seizures, and Antagonist-binding and stabilization of a co-repressor to the peroxisome proliferator-activated receptor α (PPARα) signaling complex ultimately causing starvation-like weight loss.

Validation of a Genomics-Based Hypothetical Adverse Outcome Pathway: 2,4-Dinitrotoluene Perturbs PPAR Signaling Thus Impairing Energy Metabolism and Exercise Endurance

2,4-dinitrotoluene (2,4-DNT) is a nitroaromatic used in industrial dyes and explosives manufacturing processes that is found as a contaminant in the environment. Previous studies have implicated antagonism of PPARα signaling as a principal process affected by 2,4-DNT. Here, we test the hypothesis that 2,4-DNT-induced perturbations in PPARα signaling and resultant downstream deficits in energy metabolism, especially from lipids, cause organism-level impacts on exercise endurance. PPAR nuclear activation bioassays demonstrated inhibition of PPARα signaling by 2,4-DNT whereas PPARγ signaling increased. PPARα (-/-) and wild-type (WT) female mice were exposed for 14 days to vehicle or 2,4-DNT (134 mg/kg/day) and performed a forced swim to exhaustion 1 day after the last dose. 2,4-DNT significantly decreased body weights and swim times in WTs, but effects were significantly mitigated in PPARα (-/-) mice. 2,4-DNT decreased transcript expression for genes downstream in the PPARα signaling pathway, principally genes involved in fatty acid transport. Results indicate that PPARγ signaling increased resulting in enhanced cycling of lipid and carbohydrate substrates into glycolytic/gluconeogenic pathways favoring energy production versus storage in 2,4-DNT-exposed WT and PPARα (-/-) mice. PPARα (-/-) mice appear to have compensated for the loss of PPARα by shifting energy metabolism to PPARα-independent pathways resulting in lower sensitivity to 2,4-DNT when compared with WT mice. Our results validate 2,4-DNT-induced perturbation of PPARα signaling as the molecular initiating event for impaired energy metabolism, weight loss, and decreased exercise performance.

A Systems Toxicology Approach to Elucidate the Mechanisms Involved in RDX Species-Specific Sensitivity

Interspecies uncertainty factors in ecological risk assessment provide conservative estimates of risk where limited or no toxicity data is available. We quantitatively examined the validity of interspecies uncertainty factors by comparing the responses of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) to the energetic compound 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), a known neurotoxicant. Relative toxicity was measured through transcriptional, morphological, and behavioral end points in zebrafish and fathead minnow fry exposed for 96 h to RDX concentrations ranging from 0.9 to 27.7 mg/L. Spinal deformities and lethality occurred at 1.8 and 3.5 mg/L RDX respectively for fathead minnow and at 13.8 and 27.7 mg/L for zebrafish, indicating that zebrafish have an 8-fold greater tolerance for RDX than fathead minnow fry. The number and magnitude of differentially expressed transcripts increased with increasing RDX concentration for both species. Differentially expressed genes were enriched in functions related to neurological disease, oxidative-stress, acute-phase response, vitamin/mineral metabolism and skeletal/muscular disorders. Decreased expression of collagen-coding transcripts were associated with spinal deformity and likely involved in sensitivity to RDX. Our work provides a mechanistic explanation for species-specific sensitivity to RDX where zebrafish responded at lower concentrations with greater numbers of functions related to RDX tolerance than fathead minnow. While the 10-fold interspecies uncertainty factor does provide a reasonable cross-species estimate of toxicity in the present study, the observation that the responses between ZF and FHM are markedly different does initiate a call for concern regarding establishment of broad ecotoxicological conclusions based on model species such as zebrafish.

A new approach to construct pathway connected networks and its application in dose responsive gene expression profiles of rat liver regulated by 2,4DNT.

BackgroundMilitary and industrial activities have lead to reported release of 2,4-dinitrotoluene (2,4DNT) into soil, groundwater or surface water. It has been reported that 2,4DNT can induce toxic effects on humans and other organisms. However the mechanism of 2,4DNT induced toxicity is still unclear. Although a series of methods for gene network construction have been developed, few instances of applying such technology to generate pathway connected networks have been reported.ResultsMicroarray analyses were conducted using liver tissue of rats collected 24h after exposure to a single oral gavage with one of five concentrations of 2,4DNT. We observed a strong dose response of differentially expressed genes after 2,4DNT treatment. The most affected pathways included: long term depression, breast cancer regulation by stathmin1, WNT Signaling; and PI3K signaling pathways. In addition, we propose a new approach to construct pathway connected networks regulated by 2,4DNT. We also observed clear dose response pathway networks regulated by 2,4DNT.ConclusionsWe developed a new method for constructing pathway connected networks. This new method was successfully applied to microarray data from liver tissue of 2,4DNT exposed animals and resulted in the identification of unique dose responsive biomarkers in regards to affected pathways.

Genomic investigation of year-long and multigenerational exposures of fathead minnow to the munitions compound RDX.

We assessed the impacts of exposure to an environmentally representative concentration (0.83 mg/L) of the explosive cyclotrimethylenetrinitramine (RDX) on fathead minnows (Pimephales promelas) in one-year and multigenerational bioassays. In the one-year bioassay, impacts were assessed by statistical comparisons of females from breeding groups reared in control or RDX-exposure conditions. The RDX had no significant effect on gonadosomatic index or condition factor assayed at 1 d and at one, three, six, nine, and 12 months. The liver-somatic index was significantly increased versus controls only at the 12-month timepoint. RDX had no significant effect on live-prey capture rates, egg production, or fertilization. RDX caused minimal differential-transcript expression with no consistent discernable effect on gene-functional categories for either brain or liver tissues in the one-year exposure. In the multigenerational assay, the effects of acute (96 h) exposure to RDX were compared in fish reared to the F(2) generation in either control or RDX-exposure conditions. Enrichment of gene functions including neuroexcitatory glutamate metabolism, sensory signaling, and neurological development were observed comparing control-reared and RDX-reared fish. Our results indicated that exposure to RDX at a concentration representing the highest levels observed in the environment (0.83 mg/L) had limited impacts on genomic, individual, and population-level endpoints in fathead minnows in a one-year exposure. However, multigenerational exposures altered transcript expression related to neural development and function. Environ.

The increased toxicity of UV-degraded nitroguanidine and IMX-101 to zebrafish larvae: Evidence implicating oxidative stress

Insensitive munitions (IMs) improve soldier safety by decreasing sympathetic detonation during training and use in theatre. IMs are being increasingly deployed, although the environmental effects of IM constituents such as nitroguanidine (NQ) and IM mixture formulations such as IMX-101 remain largely unknown. In the present study, we investigated the acute (96h) toxicity of NQ and IMX-101 to zebrafish larvae (21d post-fertilization), both in the parent materials and after the materials had been irradiated with environmentally-relevant levels of ultraviolet (UV) light. The UV-treatment increased the toxicity of NQ by 17-fold (LC50 decreased from 1323mg/L to 77.2mg/L). Similarly, UV-treatment increased the toxicity of IMX-101 by nearly two fold (LC50 decreased from 131.3 to 67.6mg/L). To gain insight into the cause(s) of the observed UV-enhanced toxicity of the IMs, comparative molecular responses to parent and UV-treated IMs were assessed using microarray-based global transcript expression assays. Both gene set enrichment analysis (GSEA) and differential transcript expression analysis coupled with pathway and annotation cluster enrichment were conducted to provide functional interpretations of expression results and hypothetical modes of toxicity. The parent NQ exposure caused significant enrichment of functions related to immune responses and proteasome-mediated protein metabolism occurring primarily at low, sublethal exposure levels (5.5 and 45.6mg/L). Enriched functions in the IMX-101 exposure were indicative of increased xenobiotic metabolism, oxidative stress mitigation, protein degradation, and anti-inflammatory responses, each of which displayed predominantly positive concentration-response relationships. UV-treated NQ had a fundamentally different transcriptomic expression profile relative to parent NQ causing positive concentration-response relationships for genes involved in oxidative-stress mitigation pathways and inhibited expression of multiple cadherins that facilitate zebrafish neurological and retinal development. Transcriptomic profiles were similar between UV-treated versus parent IMX-101 exposures. However, more significant and diverse enrichment as well as greater magnitudes of differential expression for oxidative stress responses were observed in UV-treated IMX-101 exposures. Further, transcriptomics indicated potential for cytokine signaling suppression providing potential connections between oxidative stress and anti-inflammatory responses. Given the overall results, we hypothesize that the increased toxicity of UV-irradiated NQ and the IMX-101 mixture result from breakdown products with elevated potential to elicit oxidative stress.