Extraction equilibria of ammonia with acidic organophosphorus compounds

Extraction equilibrium of ammonia from aqueous ammonium nitrate-ammonia mixture by various acidic organophosphorus extractants was investigated at 30°C in terms of the distribution of ammonia between two phases, water content in the organic phase and viscosity of the organic phase. The acidic organophosphorus extractants used were di(2-ethylhexyl)phosphoric acid, 2-ethylhexyl 2-ethylhexylphosphonic acid and di(2,4,4′-trimethylpentyl)phosphinic acid; toluene was used as a diluent.It was found that ammonia is extracted into the organic phase not only through the chemical interaction with the extractants but also through its physical partition into the diluent itself.The distribution through the chemical interaction with the extractants was quantitatively interpreted by the formation of the ion-pair complex, NH₄R(HR)₄, in the low concentration region of ammonia in the organic phase. In the higher concentration region, it was qualitatively interpreted by the transformation of this complex into other complexes of the types NH₄R(HR) or NH₄R. They were inferred to exist as micelles from the experimental results that the water content and viscosity largely increase in the high pH region.     

Antibody-coated sheep erythrocytes suppress the ability of polyclonal B-cell activators to induce plaque-forming cells against sheep erythrocytes

The primary immune response to untreated sheep erythrocytes (SRBC) in vitro was suppressed by the addition of antibody-coated SRBC. A mixture of SRBC and antibody-coated SRBC also suppressed the induction of anti-SRBC plaque-forming cells by the polyclonal B cell activators lipopolysaccharide, purified protein derivative of tuberculin, and native dextran. Injection of a mixture of SRBC and antibody-coated SRBC into mice led to an increased response to SRBC. It seems plausible from the in vitro findings that the Fc part of antibodies complexed to an antigen can exert a negative signal on antigen-specific B cells that cannot be overcome by positive signals delivered by polyclonal B cell activators.     

Lipoproteins and hydrolysis of organophosphorus compounds

Paraoxonase of human and animal sera was shown to be a structural part of high density lipoproteins (HDL) by immunoprecipitation, heparin- or polyethyleneglycol fractionation, ultracentrifugation and gel chromatography. Frequency distribution of paraoxonase activity in human sera is trimodal. Human individuals, with respect to paraoxon detoxication, can be distinguished into low and high detoxicators using ratios of phenylacetate and paraoxon hydrolysis as well as activation with ethanolamine and sodium chloride. With conversion of alpha-lipoprotein subtype HDL3 to HDL2, specific activities of paraoxonase and arylesterase are increasing about 3.5-fold in low detoxicator individuals and 1.9-fold in high detoxicators, indicating that more than 90% of HDL2 particle-bound paraoxonase and arylesterase activity are incorporated during the HDL conversion process. HDL cholesterol concentrations in individual sera were shown to be positively correlated to both serum paraoxonase and arylesterase activities.     

Amplification of the effectiveness of acetylcholinesterase for detoxification of organophosphorus compounds by bis-quaternary oximes

Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase (FBS AChE) provides complete protection against 5 LD50 of organophosphate (OP) without any signs of toxicity or performance decrements as measured by serial probe recognition tests or primate equilibrium platform performance (Maxwell et al., Toxicol Appl Pharmacol 115: 44-49, 1992; Wolfe et al., Toxicol Appl Pharmacol 117: 189-193, 1992). Although such use of enzyme as a single pretreatment drug for OP toxicity is sufficient to provide complete protection, a relatively large (stoichiometric) amount of enzyme was required in vivo to neutralize OP. To improve the efficacy of cholinesterases as pretreatment drugs, we have developed an approach in which the catalytic activity of OP-inhibited FBS AChE was rapidly and continuously restored, thus detoxifying the OP and minimizing enzyme aging by having sufficient amounts of appropriate oxime present. The efficacy of FBS AChE to detoxify several OPs was amplified by addition of bis-quaternary oximes, particularly 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxyaminopyridinium) -dimethyl ether hydrochloride (HI-6). When mice were pretreated with sufficient amounts of FBS AChE and HI-6 and challenged with repeated doses of O-isopropyl methylphosphonofluridate (sarin), the OP was continuously detoxified so long as the molar concentration of the sarin dose was less than the molar concentration of AChE in circulation. The in vitro experiments showed that the stoichiometry of sarin:FBS AChE was higher than 3200:1 and in vivo stoichiometry with mice was as high as 57:1. Addition of HI-6 to FBS AChE as a pretreatment drug amplified the efficacy of enzyme as a scavenger of nerve agents.     

Lung lymphocytes proliferate minimally in the murine pulmonary immune response to intratracheal sheep erythrocytes

The importance of in situ lymphocyte proliferation for net accumulation of lung lymphocytes during pulmonary immune responses and in immunologic lung diseases remains uncertain. Accordingly, we studied the experimental pulmonary immune response of antigen-primed C57BL/6 mice to intratracheal challenge with the particulate antigen sheep red blood cells. Uptake of nucleotide analogs (bromodeoxyuridine in vivo and tritiated thymidine in vitro), expression of the cell activation antigens CD25 and CD69 by flow cytometry, and response to the antimitotic agent hydroxyurea (in vivo) were measured. Although many lung lymphocytes and CD4+ T cells were CD25+ and CD69+, indicating recent activation, all techniques demonstrated that lung lymphocytes proliferated minimally in vivo. Blockade of cell division by hydroxyurea administration for 24 h did not significantly decrease lung lymphocyte accumulation on Day 3 after challenge. Lung lymphocytes also proliferated minimally in vitro (even on macrophage removal and despite addition of exogenous interleukin [IL]-2 or IL-4). However, lung lymphocytes responded vigorously to mitogens (immobilized anti-CD3, phytohemagglutinin, or concanavalin A), excluding global unresponsiveness to restimulation. Thus, in this model of pulmonary immunity, accumulation of lung lymphocytes does not require local T-cell proliferation and presumably depends instead on recruitment.     

Halothane-associated enhancement of the secondary immune response to sheep erythrocytes in mice: cell transfer studies

The effect of halothane anesthesia on the humoral immune response to sheep red blood cells was studied in mice immunized twice, with a 15-day interval. On both occasions, mice were exposed to 1.5% halothane for 40 min immediately after sensitization. Halothane reexposure resulted in increased numbers of IgG-secreting cells (IgG-SC) as well as circulating 7S-serum agglutinins. To examine further whether this effect could be obtained in syngeneic recipients, adoptive transfer experiments employing spleen cells were performed. While mice receiving cells from unimmunized and anesthetized donors displayed significantly higher levels of IgG-SC, recipients of cells from normal, immunized and immunized-anesthetized donors showed a depressed response when compared to control counterparts. Besides the possibility of an enhancing effect of halothane reexposure on the humoral response, this procedure may counteract normal physiological immunoregulatory processes during the generation of the immune response.     

Dengue virus-induced cytotoxic factor suppresses immune response of mice to sheep erythrocytes

The present study was undertaken to investigate if the suppressed cell-mediated immune responses observed in dengue type 2 virus (DV)-infected mice could be due to the cytotoxic factor (CF) produced in the spleens of DV-infected mice. We have observed that CF given intravenously (i.v.) kills splenic cells and reduces the total cells in the spleen. Mice treated with CF have a significantly depressed immune response to sheep erythrocytes, viz. delayed-type-hypersensitivity as measured by footpad swelling reaction at 24 hr; Jerne's antibody plaque-forming cells in the spleen; and migration inhibition of spleen cells in presence of antigen. These findings are similar to those seen earlier in DV-infected mice.     

Biological monitoring of organophosphorus pesticide exposure among children of agricultural workers in central Washington State

Children up to 6 years of age who lived with pesticide applicators were monitored for increased risk of pesticide exposure: 48 pesticide applicator and 14 reference families were recruited from an agricultural region of Washington State in June 1995. A total of 160 spot urine samples were collected from 88 children, including repeated measures 3-7 days apart. Samples were assayed by gas chromatography flame photometric detector for dimethylphosphate metabolites. Dimethylthiophosphate (DMTP) was the dominant metabolite. DMTP levels were significantly higher in applicator children than in reference children (p = 0.015), with median concentrations of 0.021 and 0.005 microg/ml, respectively; maximum concentrations were 0.44 and 0.10 microg/ml, respectively. Percentages of detectable samples were 47% for applicator children and 27% for reference children. A marginally significant trend of increasing concentration was observed with decreasing age among applicator children (p = 0.060), and younger children within these families had significantly higher concentrations when compared to their older siblings (p = 0.040). Applicator children living less than 200 feet from an orchard were associated with higher frequency of detectable DMTP levels than nonproximal applicator children (p =0.036). These results indicate that applicator children experienced higher organophosphorus pesticide exposures than did reference children in the same community and that proximity to spraying is an important contributor to such exposures. Trends related to age suggest that child activity is an important variable for exposure. It is unlikely that any of the observed exposures posed a hazard of acute intoxication. This study points to the need for a more detailed understanding of pesticide exposure pathways for children of agricultural workers.     

Practical management of the poisoned patient

Clinical toxicology is undergoing a change in its previously perceived unscientific image. As in other medical disciplines, there is an effort to ensure that its treatments are evidence based. A successful outcome seems likely not only to rationalize the management of poisoned patients but to simplify it.     

Effects of lysozyme dimer on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunosuppressed mice

The effects of lysozyme dimer on humoral response to sheep erythrocytes (SRBC) and restoration of the response impaired by a single cyclophosphamide dose (200 mg/kg) were tested on mice. The effect of lysozyme dimer on the humoral response to SRBC in non-treated with cyclophosphamide mice was determined in relation to doses (0.2, 2, 20 or 200 micrograms/kg) and the time of the drug administration with respect to the antigen before or after SRBC immunization. Moreover, the effect of lysozyme dimer on the humoral response in cyclophosphamide-treated mice was studied depending on the dose applied and time of exposure to the drug in relation to SRBC. It has been found that lysozyme dimer potentiates the humoral response to SRBC in mice, resulting in an increased number of splenocytes producing haemolytic antibodies (PFC) and the total and 2-mercaptoethanol resistant level of anti-SRBC antibodies. A single exposure to lysozyme dimer gave the strongest stimulating action on SRBC when the doses of 2 or 20 micrograms/kg were administered 2 h prior to the antigen. The potentiating effect of the drug was reduced when it was administered 24 h before the antigen and also when single doses were as high as 200 micrograms/kg and as low as 2 micrograms/kg. Exposure to four doses of lysozyme dimer at 24 h intervals was more activating than a single injection. A strong potentiating effect on the specific response to SRBC was noted after four injections of lysozyme dimer at doses from 0.2 to 20 micrograms/kg. The effect of the drug did not depend on the time of exposure to the antigen. It has also been found that lysozyme dimer significantly reduces the suppressive effect of a high cyclophosphamide dose (200 mg/kg) on the humoral response of SRBC-immunized mice. The protective action of lysozyme dimer was dose- and time-dependent. The strongest protection was observed after three doses of 20 micrograms/kg administered prior to pharmacological immunosuppression. Reduction in the dose to 2 micrograms/kg and shorter treatment resulted in reduced protective effects. We have also found that the protective action of three doses of lysozyme dimer (2 or 20 micrograms/kg each) administered between cyclophosphamide injection and the antigen, or after antigen administration is weaker than such a treatment prior to cyclophosphamide immunosuppression.     

Delayed neuropathy and inhibition of soluble neuropathy target esterase following the administration of organophosphorus compounds to hens

BACKGROUND: Disulfide exchange reactions are catalyzed by thiol/disulfide oxidoreductases. These enzymes possess a thioredoxin fold and contain a catalytic disulfide with the sequence Cys-X-X-Cys at the N terminus of an alpha helix. Despite these similarities, the various members differ strongly in their redox potentials (-122 mV to -270 mV). Using the strong oxidant DsbA from Escherichia coli as a model system, we investigated whether the redox properties of these enzymes can be modulated rationally by exchange of the X-X dipeptide. RESULTS: The X-X dipeptide of DsbA (Cys30-Pro31-His32-Cys33) was exchanged by the dipeptides of eukaryotic protein disulfide isomerase (PDI; Gly-His), glutaredoxin (Pro-Tyr), and thioredoxin (Gly-Pro) from E. coli. All variants were less oxidizing than wild-type DsbA and their redox potentials were in the order of the related natural enzymes (DsbA > PDI > glutaredoxin > thioredoxin). The equilibrium constant between glutathione and the thioredoxin-like variant increased 1200-fold compared with wild-type DsbA. The variants also showed a strong increase in the pKa of the nucleophilic cysteine (Cys30). As for glutaredoxin and thioredoxin, the catalytic disulfide stabilized the corresponding variants while destabilizing wild-type DsbA and the PDI-like variant. CONCLUSIONS: The X-X dipeptide in the active site of thiol/disulfide oxidoreductases appears to be the main determinant of the redox properties of these enzymes. This empirical finding should be very useful for the design of new thiol/disulfide oxidoreductases with altered redox potentials and for studying the function of these enzymes in vivo.     

Role of metabolism in ethyl carbamate-induced suppression of antibody response to sheep erythrocytes in female Balb/C mice

A possible role of metabolism by cytochrome P450 (P450) in ethyl carbamate-induced suppression of the antibody response to a T-cell-dependent antigen, sheep erythrocytes (SRBCs), was investigated in female Balb/C mice. When mice were treated with ethyl carbamate intraperitoneally for 14 consecutive days at 25, 50, 100, 200 and 400 mg/kg, the antibody response was significantly suppressed from 200 mg/kg. These doses also caused a decrease in thymus weight. An acute dosing of ethyl carbamate at 1 g/kg also caused not only a significant suppression of the antibody response, but also a decrease in thymus weight. The antibody response was most likely to be the IgM antibody response, which was demonstrated in a haemagglutination study. When mice were pretreated with phenobarbital (80 mg/kg) for 3 days to induce P450 enzymes, followed by administration of ethyl carbamate intraperitoneally for 7 consecutive days, the antibody response was more suppressed than in saline-pretreated controls. Moreover, a study using aminoacetonitrile, a P450 inhibitor, showed that the antibody response suppressed by ethyl carbamate was completely recovered by the inhibitor. The present results suggest that metabolism of ethyl carbamate by P450 may be the critical pathway to produce metabolites capable of suppressing the antibody response.