Megakaryocyte expansion and macrophage infiltration in bone marrow of rats subchronically treated with MNX, N-nitroso environmental degradation product of munitions compound RDX (hexahydro- 1,3,5-trinitro-1,3,5-triazine) Sindhura Ramasahayam, Sridhar Jaligama, Sahar M. Atwa, Joshua T. Salley, Marissa Thongdy, Benny L. Blaylock and Sharon A. Meyer* ABSTRACT: Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), environmental degradation product of munitions hexahydro- 1,3,5-trinitro-1,3,5-triazine (RDX), causes seizures in rats with acute oral exposure like parent RDX. Our previous studies have additionally reported hematotoxicity with acute MNX exposure manifested as myelosuppression, anemia and splenic hemosiderosis. This study explored whether MNX administered subchronically continued to target bone marrow to elicit periph- eral blood cytopenia. Female Sprague–Dawley rats were gavaged daily for 4 or 6 weeks with 47 mg kg À1 day À1 MNX (¼ LD 50 ) or vehicle (5% dimethyl sulfoxide in corn oil) and hematological and clinical chemistry parameters, spleen weights, spleen and bone marrow histopathology and immunohistochemistry with ED1 anti-CD68 macrophage marker were evaluated 24 h after the last dose. Unexpectedly, no decrease in blood erythroid parameters was seen with subchronic MNX and convulsions and tremors ceased after 2 weeks of treatment. Toxicological effects observed were MNX-induced increases in blood granulocyte and platelet counts and in bone marrow megakaryocyte and ED1 + -macrophage density. MNX was without effect on bone marrow cellularity and picrosirius red stained/collagen fiber deposition. Spleen weight increased modestly with extramedullary hematopoiesis evident, but hemosiderin and relative red and white pulp areas were unaffected. Collectively, this study demonstrated that erythroid effects characteristic of acute MNX exposure were not evident with subchronic exposure. However, megakaryocyte proliferation in bone marrow coincident with thrombocytosis after subchronic MNX exposure suggested continued hematotoxicity, but with a qualitatively different outcome. Granulocytosis and increased bone marrow macrophages implicated an inflammatory component in MNX hematotoxicity. Copyright © 2017 John Wiley & Sons, Ltd. Keywords: military environmental contaminant; macrophages; thrombocytosis; megakaryocyte; bone marrow inflammation Introduction Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common explo- sive and highly energetic chemical used in various military and civilian applications. The US Environmental Protection Agency (USEPA) has identified RDX as a contaminant of 16 listed and 31 proposed sites of the National Priorities List (Agency for Toxic Substances and Disease Registry [ATSDR], 2012). The US Department of Defense (Murnyak et al., 2011) and USEPA (2014) consider RDX an emerging contaminant, a classification for important military chemicals that lack adequate human health standards or have evolving science and/or regulatory status. RDX undergoes environmental nitroreduction to N-nitroso derivatives by anaerobic microbes (Crocker et al ., 2006; Hawari et al., 2000; Khan et al., 2012; McCormick et al., 1981) and abiotic degradation (Williams et al., 2005). These are hexahydro-1- nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5- nitro-1,3,5-triazine and hexahydro-1,3,5-trinitroso-1,3,5-triazine. Structures for RDX and MNX are shown in Fig. 1. In addition to RDX, its N-nitroso degradation products have been detected in soil and ground water at munitions manufacturing and artillery training sites with MNX in greatest concentrations (Beller & Tiemeier, 2002; Paquet et al., 2011). Exposure to MNX may also occur because of uptake of RDX as MNX is detected as a metabo- lite in experimental animals treated with RDX (Major et al., 2007; Pan et al., 2007). Hence, understanding toxicity of MNX is impor- tant for estimating risk to human health, particularly of workers conducting remediation of RDX-contaminated sites. Several studies have documented RDX toxicity, but little is known about the toxicity of MNX. Human occupational and acci- dental exposures to RDX and acute oral exposures in rats resulted in seizures (Burdette et al., 1988; Goldberg et al., 1992; *Correspondence to: Sharon A. Meyer, PhD, Department of Toxicology, School of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Dr., Monroe, LA 71201, USA. E-mail: meyer@ulm.edu Department of Toxicology, School of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA J. Appl. Toxicol. 2017 Copyright © 2017 John Wiley & Sons, Ltd. Research article Received: 6 December 2016, Revised: 18 December 2016, Accepted: 19 December 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jat.3439