Project description:Abstract — Previous toxicological investigations of the insensitive munition (IM), 3-nitro-1,2,4-triazol-5-one (NTO), demonstrated histopathological and physiological impacts in mammalian testes. The implications of these findings for fish was unknown, therefore we investigated the effects of chronic (21 day) exposures to NTO and an NTO-containing IM formulation called IMX-101 (composed of 2,4-dinitroanisole (DNAN), nitroguanidine (NQ), and NTO) in adult male fathead minnows to assess if impacts on testes were conserved. The NTO exposure caused no significant mortality through the maximum exposure concentration (720 mg/L, measured), however NTO elicited testicular impacts causing significant asynchrony in spermatogenesis and necrosis in secondary spermatocytes at the two highest exposure concentrations (383 mg/L and 720 mg/L) and testicular degeneration at the highest exposure. Microarray-based transcriptomics analysis identified significant enrichment of steroid metabolism pathways and mTORC-signal control of spermatogonia differentiation in NTO exposures each having logical connections to observed asynchronous spermatogenesis. Additionally, NTO impaired transcriptional expression for genes supporting sperm structural and flagellar development including sperm-associated antigen 6 (Spag6). These functional transcriptomic responses are logical contributors underlying impacted reproductive physiology in NTO exposures that ultimately lead to reductions in spermatozoa. In contrast to NTO, the IMX-101 formulation elicited significant mortality at the two highest exposure concentrations of 25.2 and 50.9 mg/L (DNAN nominal + NTO measured + NQ measured). Unlike NTO and NQ, the DNAN component of the IMX-101 formulation underwent significant transformation in the 21d exposure. From previous investigations, neither NTO or NQ caused mortality in fish at >1000 mg/L suggesting that mortality in the present study arose from DNAN / DNAN-attributable transformation products. The 12.6 mg/L IMX-101 exposure caused significant sublethal impacts on testes including sperm necrosis, interstitial fibrosis, and Sertoli-like cell hyperplasia. Transcriptional profiles for IMX-101 indicated significant enrichment on multiple signaling pathways supporting spermatogenesis, mitosis / meiosis, and flagellar structure, all logically connected to observed sperm necrosis. Additionally, pronounced transcriptional increases within the PPARα-RXRα pathway, a known DNAN target, has been hypothesized to correspond to Sertoli cell hyperplasia, presumably as a compensatory response to fulfill the nurse-function of Sertoli cells during spermatogenesis. Overall, the transcriptional results indicated unique molecular responses for NTO and IMX-101. Regarding chemical hazard, NTO impacted testes and impaired spermatogenesis, but at high exposure concentrations (≥ 192 mg/L), whereas the IMX-101 formulation, elicited mortality and impacts on reproductive physiology likely caused by DNAN and its transformation products present at concentrations well below the NTO-component concentration within the IMX-101 mixture formulation. see previous row.

Project description:Abstract — Within the US military, new insensitive munitions (IMs) are rapidly replacing conventional munitions improving safety from unintended detonation. Toxicity data for IM chemicals are expanding rapidly, however IM constituents are typically deployed in mixture formulations, and very little is known about their mixture toxicology. In the present study we sought to characterize the mixture effects and toxicology of the two predominant IM formulations IMX-101 and IMX-104 in acute (48 h) larval fathead minnow (Pimephales promelas) exposures. IMX-101 consists of a mixture of 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine (NQ) while IMX-104 is composed of DNAN, NTO, and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). DNAN was the most potent constituent in IMX-101 eliciting an LC50 of 36.1 mg/L, whereas NTO and NQ did not elicit significant mortality in exposures up to 1040 and 2640 mg/L, respectively. Toxic unit calculations indicated that IMX-101 elicited toxicity representative of the component concentration of DNAN within the mixture. Toxicogenomic responses for the individual constituents of IMX-101 indicated unique transcriptional expression and functional responses characteristic of: oxidative stress, impaired energy metabolism, tissue damage and inflammatory responses in DNAN exposures; impaired steroid biosynthesis and developmental cell-signaling in NQ exposures; and altered mitogen-activated protein kinase signaling in NTO exposures. Transcriptional responses to the IMX-101 mixture were driven by the effects of DNAN where expression and functional responses were nearly identical comparing DNAN alone versus the fractional equivalent of DNAN within IMX-101. Given that each individual constituent of the IMX-101 mixture elicited unique functional responses, and NTO and NQ did not interact with DNAN within the IMX-101 mixture exposure, the overall toxicity and toxicogenomic responses within acute exposures to the IMX-101 formulation are indicative of "independent" mixture toxicology. Alternatively, in the IMX-104 exposure both DNAN and RDX were each present at concentrations sufficient to elicit lethality (RDX LC50 = 28.9 mg/L). Toxic-unit calculations for IMX-104 mixture formulation exposures indicated slight synergistic toxicity (ΣTU LC50 = 0.82, 95% confidence interval = 0.73-0.90). Unique functional responses relative to DNAN were observed in the IMX-104 exposure including responses characteristic of RDX exposure. Based on previous transcriptomics responses to acute RDX exposures in fathead minnow larvae, we hypothesize that the potentially synergistic responses within the IMX-104 mixture are related to interactive effects of each DNAN and RDX on oxidative stress mitigation pathways.

Project description:Abstract — Insensitive munitions (IMs) improve soldier safety by decreasing sympathetic detonation during training and use in theatre. 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, both in the parent IMs and in IMs irradiated with environmentally-relevant levels of ultraviolet (UV) energy. Zebrafish were exposed to control and ten concentrations of NQ ranging from 1 to 732 mg/L (measured concentrations) or seven concentrations of IMX-101 ranging from 2 to 122 mg/L (measured concentrations) of the parent material or material that had been UV-irradiated (UV-treated) at the equivalent of 24 hours of sunlight. The UV-treatment increased the toxicity of NQ by 17-fold, indicated by a decreased LC50 from 1323 mg/L (parent compound) to 77.2 mg/L. Similarly, UV-treatment increased the toxicity of IMX-101 by nearly two fold (LC50 decreased from 131.3 to 67.6 mg/L). Molecular responses to parent and UV-treated IMs were assessed using 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 45.6 mg/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 where positive concentration-response relationships were observed for genes involved in oxidative-stress mitigation pathways whereby we hypothesize that the increased toxicity of UV-treated NQ resulted from increased oxidative stress. This hypothesis was supported by transcriptomic responses indicative of oxidative-stress responses involved in zebrafish development, especially neurological development of visual systems and the brain. 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 these result, we hypothesize that the increased toxicity of UV-irradiated NQ and the IMX-101 mixture result from breakdown products with increased potential to elicit oxidative stress.

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Molecular Evaluation of Impacted Reproductive Physiology in Fathead Minnow Testes Provides Mechanistic Insights into Insensitive Munitions Toxicology (NTO)

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