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

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. Two separate microarray assays were conducted within this study. The two experiments investigated 2 unique chemical exposures in dose-series assays including: 1. NTO and 2. the IMX-101 formulation which contains a mixture of NTO, DNAN and NQ. This GEO entry represents analysis number 1. NTO: Subchronic (21 day) toxicity tests employing adult male P. promelas were conducted as static renewal assays conducted at 23 °C. Exposures were performed in 7.6 L glass fish tanks with a test solution volume of 6.0 L following ASTM general guidance. Partial water renewals were conducted thrice weekly on Mondays, Wednesdays and Fridays by removing and replacing 3.0 L of exposure solution. Two fish were exposed in each tank to NTO at 0 (control), 94, 192, 383, 720 mg/L (measured concentrations) where each experimental treatment included 5 replicate tanks. All fish surviving until the conclusion of the 21d assay were euthanized by immersion in an overdose solution of MS-222 (300 mg/L) for at least 10 minutes after cessation of movement. After euthanasia, each fish was weighed (g) and then necropsied where one testis was fixed in 10% neutral-buffered formalin for histopathological evaluation and the other fixed for transcriptomics by flash freezing in liquid nitrogen. All animal testing was conducted using methods and protocols approved by the US Army, Engineer Research and Development Center’s Institutional Animal Care and Use Committee (IACUC Protocol #EL-6009-2014-1).