J Appl Toxicol. 2017 Aug;37(8):913-921. doi: 10.1002/jat.3439.

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).

Ramasahayam S1, Jaligama S1, Atwa SM1, Salley JT1, Thongdy M1, Blaylock BL1, Meyer SA1.

1 Department of Toxicology, School of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA.



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 peripheral blood cytopenia. Female Sprague-Dawley rats were gavaged daily for 4 or 6 weeks with 47 mg kg-1  day-1 MNX (¼ LD50 ) 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.

PMID: 28138994



For a contemporary public whose quality of life is highly dependent upon anthropogenic chemicals, the most prudent public health policy to reduce risk of chemical toxicity is to prevent exposure.  For some events with extremely hazardous and immediate outcomes, higher risk from toxicity of targeted, specific remediation chemicals are tolerated for limited times.  Examples include use of pesticides to protect valuable crop products from insect infestation and of antibiotics to counter spread of bacterial epidemics.  Another example is from munitions chemicals used to counter adversarial challenges, such as extensive use of RDX for weapons deployed in World War II, the Korean War and Vietnam conflict.

RDX toxicity to humans is known from clinical symptoms, especially seizures, incurred during manufacture and field use during these conflict events.  Some symptoms have been replicated in experimental animals thus informing a mechanistic understanding.  For neurotoxicity, the key molecular event is RDX binding to and interference with post-synaptic inhibitory activity of GABAA receptor [1].  Refinement of rat-to-human extrapolation with mode of action identification of target and physiologically based pharmacokinetic modeling has supported derivation of a recent non-cancer chronic oral reference dose (RfD) of 0.07 mg/kg/d for human neurotoxicity [2].  The RfD is one criterion used to estimate acceptable clean-up levels for contaminated sites, including redevelopment of retired DoD manufacturing and pack-and-load facilities and practice firing ranges.

Knowledge of toxicity of environmental degradation products is also important for assessing risk of RDX contaminated sites.  RDX’s 3 nitramine groups are successively metabolized by nitroreductases of anaerobic soil bacteria with the mono-nitroso derivative MNX being the most abundant in groundwater [3, 4]. Nothing was known of MNX mammalian toxicity until our report of acute effects in rats [5] in which we described ED50 of 57 mg/kg for neurotoxicity, comparable to that for RDX tested in parallel.  We also observed a delayed onset hematotoxicity as mild anemia 14 days after single oral exposure.  Based upon this 14d lag and noting that N-nitroso bioreductive prodrug candidates, such as prototype tiripazamine, cause toxicity upon 1-electron reduction in poorly perfused tissues such as bone marrow (BM) [6], we hypothesized that MNX targeted hematopoietic cells via free radical toxicity.  Feasibility of this notion was supported by our observation that EPR-detectable product was produced upon anaerobic MNX metabolism by rat liver microsomes [7].  A subsequent study demonstrated MNX suppression of formation of BM myeloid and erythroid progenitor cells (CFU-GM and BFU-E, respectively) [8].

The highlighted manuscript addresses continued BM toxicity with subchronic exposure to MNX.  Notably, no convulsions or tremors were seen after the 2nd week of 47 mg/kg/d MNX; thus, hematotoxicity was the critical effect observed for subchronic exposure.  MNX exposure for 4 and 6 weeks was clearly associated with an increase in platelets and plateletcrit in blood.  Increase in platelets was associated with a twofold increase in BM megakaryocyte density.  Another hematological effect noted in this study was an increase in blood granulocytes.  Granulocytosis, in the absence of concurrent changes in lymphocytes as observed here, can be indicative of inflammation [9].  Moreover, inflammation has been shown to increase BM megakaryocytes with evidence supporting direct effects of cytokines on megakaryopoiesis [10, 11].   Increased density of ED1+ macrophages observed in the BM, but not liver, of MNX-treated rats supports an inflammatory component targeted to BM.

Increased megakaryocyte number and clustering occur during the prefibrotic phase of clinical primary myelofibrosis (PMF) [12] (recently named primary megakaryocytic granulocytic myeloproliferation, PMGM [13]) and population of BM with an excess number of megakaryocytes is associated with deposition of reticulin fibers [14]. These observations collectively argue that accumulation of megakaryocytes in hematopoietic tissues could serve as a mediator of fibrosis upon inflammation of the BM itself. In this study, macrophage infiltration of BM increased with the time of exposure to MNX, coincident with megakaryocyte proliferation. However, fibrosis was not evident with 6 weeks of MNX treatment when probed as either silver-stained reticulin fibers or Picrosirus stained- birefringence.  Other symptoms of prefibrotic PMF, extramedullary hematopoiesis and spleen enlargement, were observed. With our experimental animal study, we could attribute the latter to expansion of red pulp from morphometric assessment of histopathological sections.

The fibrosis component of PMF is elaborated by BM stromal cells [15].  To probe for MNX-induced prefibrotic events, we assessed expression by BM nucleated cells of a transcriptome subset associated with the fibrotic process (RT² Profile PCR Array Rat Fibrosis, PARN-120Z, Qiagen).  Of these, five exhibited >2-fold increase in expression with statistical significance (p<0.05, n=4) over vehicle control upon 4 weeks of MNX treatment (Figure 1).  One of these, Col1a2 codes for a Type I pro-a2 collagen subunit that assembles with pro-a1 subunits to form procollagen.  Further evidence for potential for procollagen formation was our observation of an increase in cells with fibroblast morphology in MNX-treated BM that gave cytoplasmic immunohistochemical staining with antibody to Type I collagen (mouse monoclonal, Santa Cruz COL 1A (COL-1)) (Figure 2).



Figure 1.  Volcano plot of expression of transcripts associated with fibrosis as affected by 4 wk treatment with 47 mg/kg/d MNX relative to vehicle control.  Transcript amounts were assessed with RT² PCR and ratio of MNX to control was calculated using the 2-ΔΔCT method (https://www.qiagen.com/nl/resources/resourcedetail?id=6161ebc1-f60f-4487-8c9e-9ce0c5bc3070&lang=en).  Abbreviations are: Cav1, caveolin 1; Col1a2, Type 1 collagen a2; Dcn, decorin; Itgb3, integrin subunit beta 3; Mmp2, matrix metallopeptidase 2; Plg, plasminogen; Serpine1, Plasminogen Activator Inhibitor-1.



Figure 2.  Micrographs of BM sections stained immunohisto-chemically for Type I collagen.  Mouse monoclonal anti-bovine collagen I (1:500, Santa Cruz) and signal amplification with Vectastain Elite ABC-HRP and 3,3-diaminobenzidine were used.  Arrows indicate cells with cytoplasmic staining.


Collectively, the present results and those in the highlighted manuscript offer an experimental animal response that recapitulates progression of components of the prefibrotic phase of the clinical disease primary myelofibrosis (PMF) and, importantly, predicts that fibrosis will follow with longer exposures to MNX.  With discovery of specific mutations in CD34+ hematopoietic stem cells of PMF patients [16] and evidence for clonality of the progressed disease [17, 18], causality has been interpreted consistent with a “bad luck” concept [19], i.e., that tissue stem cells are the originating cells of oncogenic clones due to increased random mutations incurred during proliferative DNA replication.  Discovery of mutated genes with dysfunction coherent with myeloproliferative phenotype, especially JAK2V617F, have identified druggable targets and potential therapies.  However, only partial resolution of PMF symptoms – those associated with hematopoietic cells, with JAK2 inhibitors has led to appreciation that the “soil”, i.e., BM stromal niche, imposes selection pressure for the myeloproliferative phenotype of mutated stem cells [15].  Mounting evidence supports inflammation as a promoter of expansion of PMF-derived CD34+ cells with more progressed behavior [16, 20].  Dominance of JAK2V617F mutated clone has been shown to occur late in progression and correlate with myelofibrosis [17], which indicates that inflammation precedes and is a likely driver of fibrogenesis.  Stimulation of megakaryopoiesis by proinflammatory cytokines [10, 11] argues that megakaryocytes are the source of fibrogenic stimulus.  The well-known storage of TGFb in megakaryocyte alpha-granules provides an abundant reservoir of fibrogenic growth factor for stimulation of inflammation-driven fibrosis [21].

Comparison of chemically induced adverse effects in experimental animals is receiving increasing resistance as predictive for an environmental component of human disease.  For the myeloproliferative diseases, recently discovered mutations in hematopoietic cells have led to attribution of etiology of the “primary” disease to intrinsic, genetic factors.  However, numerous environmental hematotoxicants, e.g., benzene, have been shown to cause an “acquired” BM dysfunction with symptoms of the primary diseases. Although clonality, as defines the primary myeloproliferative diseases, is not evident in founders of acquired disorders, recognition of synergy between the phenotype of mutated hematopoietic cells with chemically induced, non-genetic selectors of the stromal niche defends relevance of chemical toxicity to clonal human disease. Recognition of such a relationship enables conceptualization of a unifying hypothesis to understand how adverse effects of chemicals in experimental models can inform clinical disease and provides guidance for identifying contributing gene-environment interactions.



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