Biomarkers of oral exposure to 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole (DNAN) in blood and urine of rhesus macaques (Macaca mulatta).

The U.S. Department of Defense is using the chemicals 2,4-dinitroanisole (DNAN) and 3-nitro-1, 2,4-triazol-5-one (NTO) in new munitions development. In a screen for biomarkers of exposure, these compounds were measured in urine and blood of male rhesus monkeys after oral doses. NTO peaked at 4 h, with urinary concentrations at least 100-fold higher than that of blood or serum while 4-dinitrophenol (DNP), a metabolite of DNAN, appeared in blood at concentrations 10- to 20-fold higher than the parent compound. For human exposure monitoring, urine is optimal for NTO while the metabolite DNP in blood is best for DNAN.

Dynamic hollow-fibre liquid phase microextraction of dinitrophenols from human plasma: optimization of an extraction flow system using experimental design methodology.

The utility of a dynamic hollow-fibre liquid phase microextraction method (optimized using a four-variable experimental design and response surface modelling) for extracting dinitrophenolic compounds from human plasma samples was evaluated. The investigated variables were donor phase salt concentration (10-400 mM), donor phase pH (2-6), acceptor phase pH (7-12), and donor/acceptor phase flow rates (30/7.5 to 70/17.5 microL min(-1)). Four dinitrophenol pesticides were used as model substances at concentrations of 0.1 microg mL(-1) in spiked human plasma samples. Extraction efficiencies ranging from 42 to 77% with RSDs below 9 were achieved with the optimized method. The flow rate and acceptor pH were shown to strongly affect the extraction efficiency for all compounds, while the donor phase pH and salt concentration had minor effects. With a well-defined acceptor phase pH and flow rate the system exhibited high robustness. The limits of quantification for the investigated compounds, using the presented extraction method followed by liquid chromatography/electrospray ionization mass spectrometry in selected ion monitoring mode, ranged from 0.05 to 0.1 microg mL(-1) plasma.

Is the glutathione S-conjugate pump a flippase?

A hypothesis of the flippase nature of the glutathione S-conjugate transport is presented. Experimental premises for this hypothesis include interaction of glutathione S-conjugates with the membrane, as demonstrated by their effects on membrane fluidity, quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene fluorescence and induction of echinocytosis by 2,4-dinitrophenyl-S-glutathione (DNP-SG). This hypothesis can rationalize, i. a., observations of the enhancement of DNP-SG transport by butanol and stimulation of erythrocyte membrane Mg(2+)-ATPase activity by albumin-coupled DNP-SG.

Role of neutrophils and nitric oxide in lung alveolar injury from smoke inhalation.

We examined potential mechanisms responsible for the parenchymal lung injury seen in an animal model of smoke inhalation with concurrent inflammation. Rats injected with sterile glycogen and exposed to smoke generated by the nonflaming pyrolysis of combined Douglas fir wood and polyvinylchloride showed a 74% increase in 125I-albumin lung permeability and a fivefold increase in lung myeloperoxidase (MPO) compared with control rats. There was also a significant increase in plasma indices of oxidative injury in these animals. Compared with control animals, plasma concentrations of thiobarbituric acid reactive substances (TBARS) were elevated by 62%, the concentrations of reduced sulfhydryl groups declined by 37%, and the levels of dinitrophenylhydrazine-reactive proteins (DNPH-RP) were doubled. In addition, the plasma concentrations of nitrate (NO3-) in rats exposed to glycogen plus smoke were increased three times that of control animals. Injection of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), immediately after smoke exposure or induction of neutropenia using either nitrogen mustard or antineutrophil antiserum, abolished the increase in concentrations of circulating NO3-, and prevented changes in plasma concentrations of TBARS, DNPH-RP, lung MPO activity, and tissue permeability index. These data suggest that neutrophil activation and the production of nitric oxide-derived oxidants contribute to the lung and plasma indices of oxidative injury in this smoke inhalation model.

Elimination kinetics of circulating antigens and immune complexes. I. A simple multicompartment model for the analysis of blood elimination of intravenously injected antigens and immune complexes.

A multi-compartment model designed to analyse the blood elimination kinetics of intravenously injected radiolabelled antigens in experimental animals is presented. The model was fitted to experimental data on blood radioactivity levels at different times following intravenous injection of radiolabelled antigen. Numerical values were obtained for tissue association, degradation, and urinary recovery of degradation products. The model may prove useful as a test for reticuloendothelial function.

[Binding ability of serum albumin in thermal burns]. / Sviazyvaiushchaia sposobnost' syvorotochnogo al'bumina pri termicheskikh ozhogakh.

Binding ability of blood serum albumin was decreased in patients with thermic burns as well as in rats with burns. Loss of this albumin property did not depend on decrease of its content in blood serum; in the patients with burns the decrease in albumin binding ability correlated with development of encephalopathic symptoms. After treatment of blood serum from experimental animals with hemosorbent the blood serum binding ability was distinctly restored in vitro. At the same time, hemosorption did not affect the binding ability of blood plasma in patients with burns. Importance of alterations in binding ability of blood serum albumin for development of acute burns toxemia is discussed.

Plasma levels and kinetic disposition of 2,4-dinitrophenol and its metabolites 2-amino-4-nitrophenol and 4-amino-2-nitrophenol in the mouse.

Eleven groups of six ICR mice were dosed orally with 22.5 mg/kg 2,4-dinitrophenol. Groups were sacrificed at 0, 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 96 h post-treatment and plasma was collected for analysis of dinitrophenol, 2-amino-4-nitrophenol, and 4-amino-2-nitrophenol content. Analyses were performed by capillary gas chromatography--mass spectrometry after liquid--liquid extraction of plasma specimens spiked with two internal standards. Quantification was based upon peak-area ratios of base peaks obtained from the three analytes and the trideuterated internal standards 2,4-dinitrophenol and 2-amino-4-nitrophenol. Plasma concentrations for each analyte versus their respective time periods were subjected to pharmacokinetic analysis. Of the two monoamine metabolites, 2-amino-4-nitrophenol was present in the greater amount and had an elimination half-life of 46 h from plasma while that of 4-amino-2-nitrophenol was 26 h.

Dinitrophenol poisoning: a diagnosis to consider in undiagnosed fever.

A 32-year-old farmer had signs and symptoms of dinitrophenol poisoning after crop spraying with a herbicide containing derivatives of 2,4-dinitrophenol. Dinitrophenol causes toxicity by the uncoupling of oxidative phosphorylation in the mitochondria of cells throughout the body. In man the classic syndrome consists of lassitude, malaise, headache, increased perspiration, thirst, and dyspnea which may progress to hyperpyrexia, profound weight loss, respiratory failure, and death. Because dinitrophenol compounds are widely used, it is likely that some patients with unexplained fever have unrecognized dinitrophenol poisoning. Prompt recognition of the clinical manifestations of toxicity is vital for appropriate therapy. We describe a method for detection of dinitrophenol in plasma and urine.

Differential reaction of cell membrane phospholipids and proteins with chemical probes.

The major aims of this study were to determine the degree of phospholipid asymmetry and the neighbor analysis of phospholipids in different types of cell membranes. For this study a penetrating probe (FDNB), a non-penetrating probe (TNBS) and a cross-linking probe (DFDNB) were used. The reaction of hemoglobin, membrane protein and membrane PE and PS of erythrocytes with DFNB and TNBS was studied over a concentration range of 0.5 to 10 mM probe. TNBS reacts to an extremely small extend with hemoglobin over the concentration range 0.4 to 4 mM whereas FDNB reacts with hemoglobin to a very large extent (50 fold more than TNBS). The reaction of membrane protein of intact erythrocytes reaches a sharp plateau at 1 mM TNBS whereas the reaction of membrane protein goes to a much larger extent with FDNB with no plateau seen up to 4 mM FDNB. This data shows that TNBS does not significantly penetrate into the cell under our conditions whereas FDNB does penetrate into the cell. The results show that there are four fold more reactive sites on proteins localized on the inner surface of the erythrocyte membrane as compared to the outer surface. TNBS at 0.5 to 2 mM concentration does not label membrane PS and labels membrane PE to a small extent. The reaction of PE with TNBS shows an initial plateau at 2 mM probe and a second slightly higher plateau between 4 to 10 mM probe. TNBS from 0.5-2.0 mM does not react with PS, but between 3 to 10 mM concentration, a very small amount of PS reacts with TNBS. Hence above 2 mM TNBS or FDNB a perturbation occurs in the membrane such that more PE and PS are exposed and react with these probes. These results demonstrate that essentially no PS is localized on the outer surface of the membrane and only 5% of the total membrane PE is localized on the outer surface of the erythrocyte membrane. TNBS and FDNB were reacted with yeast, E. coli, and Acholeplasma cells. With yeast cells, FDNB reacts to a much larger extent with PE than does TNBS, indicating that FDNB penetrates into the cell and labels more PE molecules. With E. coli, but not with erythrocytes or yeast cells, phospholipase A activity was very pronounced at pH 8.5 giving rise to a large amount of DNP-GPE from DNP-PE. A phosphodiesterase was also present which hydrolyized DNP-GPE to DNP-ethanolamine. The multilayered structure of the E. coli cell envelop did not permit a definitive interpretation of the results. It is clear, however, that TNBS and FDNB react to a different extent with PE in this cell. The Acholeplasma membrane had no detectable PE or PS but contains amino acid esters of phosphatidylglycerol. The reaction of these components with TNBS and FDNB indicate that these aminoacyl-PG are localized on both surfaces of the membrane, with 31% being on the outer surface and 69% on the inner surface...