Free radical metabolite of munition RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and 1-nitroso degradation product MNX determined with electron paramagnetic resonance (EPR) and study the toxic effects of deepwater horizon crude oil in rats.

摘要

RDX and its N-nitroso environmental degradation product, MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), are ground water and soil contaminants of retired DoD munitions plants. Previously, researchers from our laboratory have demonstrated that RDX and MNX cause delayed-onset anemia and granulocytopenia due to the loss of bone marrow progenitor cells after a single oral dose (94 mg/kg) in rats. Myelosuppression of N-nitroso bioreductive prodrugs, (e.g., prototype tirapazamine), is thought due to reductive metabolism to free radical in hypoxic bone marrow niches. In this study, we hypothesize that RDX and MNX undergo reductive metabolism to free radical(s) that are potential mediators of the observed hematotoxicity.;Methods: RDX and MNX (50 microM) were incubated with female SD rat liver microsomes (0.25 mg/ml in 100 mM phosphate buffer and 5mM magnesium chloride, pH 7.4) and NADPH (0.5 mM) under aerobic and anaerobic conditions (N2 bubbling for 15 min). Samples were quenched at 0-60 min and run on RP (C18)-HPLC to determine the loss of RDX and MNX. Electron paramagnetic resonance (EPR) spectroscopy with spin trap 4-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN, 10mM, Tris-HCl 10mM pH 7.4) was used to detect free radicals. Incubation under anaerobic conditions led to metabolism with rates of loss of RDX approximately three times greater than for MNX. Free radical(s) were detected and levels were comparable for RDX and MNX. Metabolism of RDX and MNX to more polar product(s) and free radical(s) was NADPH-dependent. Negligible RDX and MNX loss and free radical production were occurred under aerobic conditions. We conclude that both RDX and MNX are reduced by microsomal NADPH dependent reductase(s) to free radicals that have potential to mediate observed hematotoxicity.;The second project was focused on the health effects of crude oil, which are generally estimated from previously reported effects of oil spills. However, the compositional differences from different sources indicate that the effects of each spill would vary. In this research, we identified the toxic effects of exposure to crude oil released from the Deep-Water Horizon accident. The crude oils obtained from British Petroleum (BP) were A010G4 "Surrogate" (G4), A0083Q "MASS Aug 15" (3Q), A001EQ "SOB MAY 22" (EQ) and Z00377 "OFS Juniper" (77), where the latter being a sample of surface "mousse". EPR was used to detect free radicals in these crude oil samples.;To estimate the toxicity of each sample, female Sprague-Dawley rats were treated with two doses of oils (2.5, 5 ml/kg, p.o.) daily and were then bled 48 hr later for hematological tests, clinical chemistry assays, and assay for oxidative stress using biomarker 8-isoprostane. Bone marrow cells were assayed for myeloid progenitors as well (CFU-GMs).;The results show that the exposure to all oils resulted in elevated serum alkaline phosphatase levels. Also, liver weight and CYP1A1 protein were increased with all DWH crude oils, but 3Q oil had the most notable effect in this regard. Granulocytes were observed to increase in all treated rats. However, this effect was significant only with high doses of oil in cases of 3Q and EQ oils. CFU-GMs were also increased in all exposed rats, but significant increases were observed only with the high dose of oil 77, 3Q, and a low dose of EQ oil. Serum 8-isoprostane oxidative stress biomarker showed significant increase with all DWH crude oils except for a low dose of oil 77 and high dose of G4. An asphaltene free radical spectrum was detected by EPR for all oils and the greatest intensity was observed with G4 DWH oil.;The results obtained in this research are consistent with known effects of crude oil constituents but also suggest that differing amounts, sources, and types determine the extent of toxicity that different crude oils may have.