Specific soman-hydrolyzing enzyme activity in a clonal neuronal cell culture.

An enzymatic activity that specifically hydrolyzes the highly toxic organophosphorus anticholinesterase compound soman (pinacolyl methylphosphonofluoridate) has been identified and partially characterized in the clonal neuronal neuroblastoma-glioma hybrid NG108-15 cell line. Using the whole cell homogenate as the enzyme source and 1 mM substrate, the relative rate of hydrolysis of two other toxic anticholinesterase compounds sarin (isopropyl methylphosphonofluoridate) and tabun (ethyl-N-dimethyl phosphoramidocyanidate) is approximately one-tenth the rate of hydrolysis of soman, while DFP (diisopropyl phosphorofluoridate), paraoxon (p-nitrophenyl diethylphosphate), and a phosphinate PNMPP (p-nitrophenyl methyl (phenyl) phosphinate) are not hydrolyzed. Analysis of the kinetics of soman hydrolysis reveals two components of the enzyme activity with different affinities and reaction rates. Unlike previously reported enzymes of this type, this enzyme lacks chiral specificity and thus hydrolyzes both toxic and non-toxic soman stereoisomers at equal rates. The enzyme activity is stable at low temperature, found almost exclusively in the soluble fraction of these cells, and enhanced significantly by Mn2+ and by chemical differentiation of these cells in culture. The results suggest possible application of this enzyme for soman detection and/or detoxication, and use of the NG108-15 cell line to study the natural function(s) of enzymes of this type.

In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger.

Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.

Antibody quantitation using an immunoadsorbent and the unlabeled antibody enzyme method.

Measurement of specific immunoadsorbent-bound antibodies has been accomplished by the unlabeled antibody enzyme method (Sternberger et al., 1970). Sepharose-4B containing specific antigen (or ligand) is treated with diluted specific immune serum (primary serum), such as 1 ml of serum diluted 6000-20,000 fold. followed by antiserum against immunoglobulin G (IgG) of the primary serum and then by peroxidase-antiperoxidase (antigen-antibody) complex (PAP) derived from the same species as the primary serum. Radiolabeled primary antibody and anti-IgG have confirmed the stoichiometry of the reaction. The immunoabsorbent binds the antibody of interest quantitatively and to equal extent after 15 min or 48 h. The enzymatic activity of the PAP complex followed a direct linear relationship to its concentration indicating the stability of binding in the PAP complex. A direct relation between the enzymatic activity measured when both the primary antiserum and the anti-IgG are used allows for quantitation of the antibody level of the primary serum.

Monoclonal antibodies against soman: characterization of soman stereoisomers.

Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman (C+ P+, C+ P-, C-P+ and C-P-). In all cases the antibodies tested exhibited IC50 values of 10(-4)-5 x 10(-6) M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-soman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers.

Hepatic subcellular localization of cresylbenzodioxaphosphorin oxide (CBDP)-sensitive soman binding sites.

The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.

Partial characterization of an enzyme that hydrolyzes sarin, soman, tabun, and diisopropyl phosphorofluoridate (DFP).

The properties of a rat liver enzyme that hydrolyzes organophosphorus (OP) inhibitors of cholinesterases were studied. The rates of hydrolysis of OP inhibitors were determined by continuous titration of released hydrogen ions, using a pH stat method. Centrifugation of homogenates at 205,000 g for 30 min demonstrated that the activity was in the soluble fraction. Hydrolysis of sarin, soman, and diisopropyl phosphorofluoridate (DFP), but not of tabun, was stimulated by the addition of Mn2+ and Mg2+. Hydrolysis of sarin greater than soman greater than tabun greater than DFP. Unlike other OP hydrolases that preferentially hydrolyze the non-toxic isomers of soman, this enzyme hydrolyzed all four soman isomers at approximately the same rate. This result was obtained in vitro by gas chromatographic analysis of enzyme-catalyzed soman hydrolysis and confirmed in vivo by demonstrating reduced toxicity in mice of soman partially hydrolyzed by this enzyme. Km and Vmax were determined by fitting V vs [S] to a hyperbolic function using regression analysis. Km values ranged from 1.1 mM for soman to 8.9 mM for tabun. Vmax values ranged from 54 nmol/min/mg protein for DFP to 2694 for sarin. The enzyme was stable for at least 2 months at -90 degrees but was inactivated by heating at 100 degrees for 5 min. Elution profiles from gel filtration by high pressure liquid chromatography showed that the hydrolytic activity for the OP inhibitors eluted in a single peak, suggesting that a single enzyme was responsible for the observed hydrolysis. Further purification and characterization of this enzyme should prove useful for the development of methods for detection, detoxification, and decontamination of these cholinesterase inhibitors.

Effects of repeated injection of sublethal doses of soman on behavior and on brain acetylcholine and choline concentrations in the rat.

The effects of repeated exposure to a sublethal dose (60 micrograms/kg; 0.4 LD50) of soman on brain regional acetylcholine (ACh) and choline (Ch) levels, spinal cord cholinesterase (ChE) activity and on water consumption, body weight and gross behavioral changes were examined. Male rats were dosed once a week or three times a week and at 24 h after 2, 4 or 6 weeks of dosing, selected brain tissues and behavior were examined. During the 6-week period, there was no difference between control and soman-dosed rats in water consumption or body weight under either treatment regimen. The animals treated once a week adapted to this exposure regimen well. They exhibited no change in the levels of ACh or Ch in any of the brain areas when examined at the end of 2, 4 or 6 weeks, nor did they show any obvious signs of poisoning. The total ChE activity fluctuated between 70 and 100% of control. When treated three times a week, however, survivors (90%) of the soman-treated rats developed signs that progressed in severity to a hyper-reactivity syndrome which consisted of an exaggerated reaction to mild tactile stimuli. Brain ACh levels did not change and ChE activity showed inhibition of 40, 58 and 75% when measured at 2, 4 and 6 weeks, respectively. At the end of 6 weeks, the levels of Ch, except in the striatum, were significantly elevated in brainstem, cerebral cortex, hippocampus, midbrain, and cerebellum (52%, 147%, 68%, 46%, and 91%, respectively), indicating that Ch metabolism in neuronal membranes may be altered following more frequent low-dose soman exposures.

Detection and quantification of the organophosphate insecticide paraoxon by competitive inhibition enzyme immunoassay.

A competitive inhibition enzyme immunoassay (EIA) was developed to detect and quantify levels of the organophosphate insecticide paraoxon in body fluids. Protein-conjugated paraoxon served as an immunogen for the production of rabbit heteroantiserum, from which affinity purified IgG anti-paraoxon antibodies were isolated using a heterologous protein-paraoxon-conjugated immunoabsorbent. In the competitive inhibition EIA a standard curve was generated for the inhibition of binding of anti-paraoxon IgG to a solid-phase bound heterologous protein-paraoxon conjugate by various concentrations of free paraoxon. Binding was proportionate to the color change of an appropriate substrate generated by an enzyme-conjugated second antibody specific for the rabbit IgG anti-paraoxon. The assay detected paraoxon levels as low as 10(-10)M (28 pg/ml) in buffer, and serum paraoxon levels as low as 10(-9)M. In addition to its sensitivity, this technique is ideally suited to the simultaneous processing of large numbers of samples in less than 2 hr. The competitive inhibition EIA is cost effective and should facilitate environmental surveillance using sentinel animals, expand laboratory toxicology studies, and improve clinical detection capabilities.

Kinetic properties of soluble and membrane-bound acetylcholinesterase from electric eel.

Using electric eel acetylcholinesterase (AChE) which was either membrane-bound (AChEm) or solubilized (AChEs), similar kinetics were seen in the absence of inhibitor or in the presence of edrophonium, trimethylammonium ion or paraoxon. Thus, both forms of the enzyme appear to behave similarly toward various inhibitors. However, in the presence of a probe sensitive to allosteric effects or changes in membrane fluidity, the two forms exhibit altered behavior. In the presence of F-, the relative rate of substrate hydrolysis by AChEm was reduced more rapidly than with AChEs, whether or not paraoxon was present. When inhibition by paraoxon (10(-7)-10(-4) M) was studied in the presence of F-, AChEs had a Hill coefficient of 1.0, whereas with AChEm the Hill coefficient changed from 0.8 to 1.5.

Catalytic antibodies hydrolysing organophosphorus esters.

Transition state stabilization is considered one means by which enzymes reduce free energy of activation. The transition state of phosphonic acid anhydrides acted on by OPA hydrolase is postulated to be pentacoordinate, which ordains either a square pyramid or a trigonal bipyramid structure. The advent of catalytic monoclonal antibodies has provided a system in which these assumptions can be tested. By immunizing mice with the protein conjugate of a trigonal bipyramid transition state analog, we have produced hybridomas secreting monoclonal antibodies which hydrolyze phosphonates. To date, activity has been shown toward pinacolyl methylphosphonofluoridic acid (soman). Preliminary results suggest that the antibody is an IgG2a with kappa light chain character. Our results support the trigonal bipyramidal transition state for this group of enzymes.

Organophosphate skin decontamination using immobilized enzymes.

We previously demonstrated that a combination of cholinesterase (ChE) pre-treatment with an oxime is an effective measure against soman and sarin. We describe here a novel approach for the preparation of covalently linked ChEs which are immobilized to a polyurethane matrix. Such preparation of ChE-sponges enhances the stability and usefulness of the enzymes in non-physiological environments. The ChE-sponges, which can be molded to any form, can effectively be used to remove and decontaminate organophosphates (OPs) from surfaces, biological (skin or wounds) or otherwise (clothing or sensitive medical equipment), or the environment. The ChE-sponges retained their catalytic activity under conditions of temperature, time, and drying where the native soluble enzyme would rapidly denature, and can be reused in conjunction with oximes many times. The ChE-sponge in the presence of oxime repeatedly detoxified OPs such as DFP or MEPQ. These developments in ChE technology have extended the applicability of OP scavengers from in vivo protection, to a variety of external detoxification and decontamination schemes. In addition to treatment of OP-contaminated soldiers, the ChE-sponge could protect medical personnel from secondary contamination while attending chemical casualties, and civilians exposed to pesticides or highly toxic nerve agents such as sarin.

The effects of soman, in vivo and in vitro, on aldolase activity.

We examined the in vivo and in vitro effects of soman on aldolase activity. Male rats were killed at 4, 6, 9 and 12.5 min after subcutaneous (s.c.) administration of 90 micrograms/kg soman. At 4 min after treatment, aldolase activity was inhibited 55-90% compared to control in cerebral cortex, brainstem, mid-brain and cerebellum; and up to 50% of control in diaphragm and muscle. By 12.5 min, aldolase activity in all areas had returned to control levels except in the diaphragm, which still exhibited 36% inhibition. In vitro activity of rabbit muscle aldolase was not inhibited by 10(-3) M soman. In contrast, diisopropylfluorophosphonate (DFP, 10(-3)-10(-2) M) acted as a competitive inhibitor of aldolase activity in vitro. The results suggest that in vivo, soman effects on aldolase activity are transitory. Any long-lasting effects of soman on aldolase activity may occur only in the periphery, and not in the central nervous system.

A pharmacodynamic model for soman in the rat.

A pharmacodynamic model for inhibition of acetylcholinesterase (AChE) by soman was developed to describe the intertissue differences in AChE inhibition, the dose response of AChE to inhibition by soman, and the effect of differences in xenobiotic metabolism on soman toxicity. Based on the principles of physiological pharmacokinetics, this pharmacodynamic model consisted of a set of mass balance equations that included parameters for blood flow, tissue volumes, soman metabolism, tissue/plasma partition coefficients, initial AChE levels, and the rate constant for AChE inhibition. Sensitivity analysis of the model revealed that variation of the soman metabolism parameter in plasma was the most important determinant of variation in the inhibition of brain AChE by soman.

Transport of cyanide into guinea pig cardiac mitochondria.

The transport of cyanide (CN) into cells has been presumed to be by passive diffusion. Recently, there have been reports that CN, in the form of an anion, may enter the cell by active or facilitated transport. To characterize the mechanism(s) and kinetics of CN movement across the cell membrane, we measured the rate of 14CN (Na salt) uptake into guinea-pig mitochondria. Initial velocities of CN movement into mitochondria were determined at time points ranging from 10-100 msec and at CN concentrations ranging from 1 microM-10 mM using a rapid filtration device. A Hofstee plot of the data suggests that an active or facilitated transport predominates at lower CN concentrations (less than 10 microM), whereas passive diffusion of CN predominates at higher CN concentrations. The kinetic constants for the active phase transport were Jmax = 0.9 pmol/ms and Kt = 14 microM. These results suggest that a large portion of CN movement across the cell membrane is due to an active or facilitated transport phenomenon.

Behavioral decrements persist in rhesus monkeys trained on a serial probe recognition task despite protection against soman lethality by butyrylcholinesterase.

Recently, it has been demonstrated that an exogenously administered enzyme such as butyrylcholinesterase (BuChE) can prevent death in rhesus monkeys exposed to multiple-lethal doses of the acetylcholinesterase inhibitor soman when the enzyme is given prior to soman exposure (3). We report that despite BuChE protecting against soman-induced lethality, behavioral effects are seen in these monkeys which last for at least 6 days as measured by performance on a serial probe recognition (SPR) task. Analyses of the serial position curves showed that performance was lower on the probe trials when the probe items were from the middle of the list than when the probe items were from the beginning or end of the list which were unaffected. BuChE given alone also produced behavioral effects, causing all animals not to respond on the probe trials until 8 h following BuChE administration. Taken together, these findings suggest that the BuChE is not completely binding all of the soman and that a concentration of soman which is capable of causing behavioral effects may be entering the CNS.

Effect of an anticholinesterase compound on the ultrastructure and function of the rat blood-brain barrier: a review and experiment.

Soman, an organophosphorous irreversible inhibitor of acetylcholinesterase, was studied for its effect on the rat blood-brain barrier (BBB) during the first 24 h of intoxication. Young adult male Sprague-Dawley rats, injected with Evans blue-dye and surviving a subsequent single convulsive dose of soman (114 micrograms/kg, 0.9LD50), presented focal and diffuse penetration of dye in areas of brain normally considered protected by the BBB. Invasion was widest during the first hour when signs of excitation, respiratory distress and convulsions peaked and was absent at 24 h. During this time period, cholinesterase inhibition, as measured by enzyme assay, persisted in brain and blood at 10% and 6% of control values respectively. Brains of nonconvulsing animals and animals pretreated with nembutal (45 mg/kg, I.P.) or with diazepam (10 mg/kg, I.P.) were free of extravasated dye. A ranking of dye-breached brain areas suggested that cerebellar and cerebral cortex were most frequently involved while brain stem was rarely stained. Ultrastructural analysis of breached areas with horseradish peroxidase as a tracer molecule, revealed that the probable subcellular mechanism of the induced breach was enhanced vesicular transport, a mechanism similarly described for seizure. Consequences of the breach were emphasized with the detection of significantly elevated levels of an exogenously administered quaternary compound, 3H-hexamethonium. These findings present additional evidence that an anticholinesterase-induced breach of the rat blood-brain barrier is convulsive dependent, demonstrates BBB mechanisms similar to that of seizure, and can allow CNS penetration of blood-borne drugs and circulatory proteins that normally would be slowed or excluded by an intact BBB.(ABSTRACT TRUNCATED AT 250 WORDS)