Studies on toxic effects of environmental contaminants hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and crude oil in rats

Hexahydro-l-nitroso-3,5-dinitro-1,3,5-triazine (MNX), an environmental nitro reduced product of munitions RDX, is found as a contaminant in military sites. Acute exposure to MNX and the parent RDX showed incidence of seizures in rats and humans. Earlier studies in our laboratory identified hematotoxic effects of MNX in the form of a mild decrease in blood hemoglobin content and about ~50% loss in granulocytes after single oral exposure at 14 days (NOAEL=47 mg/kg). Our earlier studies showed that loss of blood granulocytes and erythrocytes is due to delayed loss of bone marrow myeloid and erythroid progenitor cells. However, the mechanism causing delayed myelosuppressive effects is unknown. The primary objective of the present study is to address whether delayed effects of MNX are due to persistence of early hematological effects or are late-onset due to a required expression period. Female Sprague-Dawley rats were orally gavaged with MNX from (0-94 mg/kg) and hematology, organ weights, and tissue histopathology were evaluated at different time points. Early splenic effects were observed in the form of decreased relative spleen weight at 48 h, increased infiltration of activated macrophages at 48 hr (≥ 47 mg/kg) and splenic hemosiderosis at 48 hr (≥ 47 mg/kg), 7 days ((≥ 47 mg/kg) and 14 days (NOAEL= 47 mg/kg). Blood granulocytes and serum inflammatory cytokine RANTES were significantly increased at 24 hr and 48 hr after exposure. Immunohistochemistry (IHC) of MNX (94 mg/kg)-treated rat iliums (24 hr, 48 hr and 10 days) and flow cytometric analysis of BM cells (48hr and 10 days) showed increased macrophage infiltration. Increased clonal CFU-F colonies composed of endothelial cells, fibroblasts and macrophages, were obtained from BM stromal cells of MNX-treated rats. Gene expression analysis showed up regulation of NFkappaB signaling pathway in BM cells at 10 days. Together, these observations indicated inflammation in the BM early after MNX exposure that increased with time and persisted through 14 days. Collectively these results support a role for preceding and concurrent inflammation in causation of myelosuppression thus accounting for its 10 day delay in onset.;A second set of studies focused upon myelosuppressive effects of crude oils. Crude oil varies with source in its relative composition of alkanes, simple and polycyclic aromatics, nitrogen, sulfur, asphaltene and porphyrin nickel and vanadium. Here, we identify several toxicological effects upon acute exposure of rats to crude oil from several sources and correlate these with peaks of select constituents from spectra of the crude oils obtained with EPR and 1H- and 13C-NMR. Sources of oil were Louisiana sweet crude, Nigerian (Qua Iboe) sweet crude and Iraq high sulfur (ONTA, Inc., Toronto, Canada). Female Sprague-Dawley rats were given 2 daily doses of 2.5 and 5 ml/kg of oil or vehicle (0.5% DMSO in corn oil) by oral gavage. Rats were euthanized after 48 hr and blood was taken for hematology, clinical chemistry and immunoassay for vasoactive agents. Femur bone marrow cells were assayed for CFU-GM myeloid progenitors. Nigerian and Iraqi, but not LA sweet crude, oils elevated serum alkaline phosphatase (ALP), in agreement with liver pathology. Liver weight increased 25-75% and CYP1A1 protein was elevated with all, Iraqi oil being most effective. Granulocytes were increased ~2-fold by high dose of LA sweet crude oil. Spleen weights decreased 30% with high dose of Iraqi and Nigerian oil. CFU-GMs decreased (40%) only with high dose of LA sweet crude oil. Vasoconstrictor endothelin-1 increased in serum from rats treated with high dose of all oils, while NO decreased with both doses of all oils. 1H- and 13C-NMR spectra gave qualitatively similar alkane peaks for all oils, but differ in aromatic peaks, e.g., benzene (1H-NMR 7.38 ppm) of Nigerian oil was least intense. EPR spectra of all oils exhibited asphaltene free radical peak with intensity greatest for Iraqi oil. Vanadium porphyrin was detected in Iraqi oil only. These results are consistent with the previously described effects of crude oil constituents on myelosuppression, vascular endothelial cell dysfunction, hepato- and immunotoxicity, and liver metabolic enzyme induction in rodents and humans. However, the extent of these effects varied markedly among the different oil sources. Summarizing our observations, LA sweet crude oil was uniquely myelosuppressive but with lesser effects on spleen and liver weights and ALP. Myelosuppression was correlated with benzene content of the crude oils. Iraqi oil, richest in asphaltene free radical, vanadium porphyrin and PAHs, most affected liver weight and CYP1A1.