Degradation of nerve agents by an organophosphate-degrading agent (OpdA).

Enzyme-catalysed degradation of the nerve agents tabun, sarin, ethyl sarin and soman by three variants of an organophosphate-degrading enzyme was studied at low concentrations of nerve agent. The concentration of nerve agent at a given time was determined by its ability to inhibit the enzyme acetylcholinesterase. Experiments were conducted in 96-well microtitre plates. Values of the ratio of k(cat) (turnover number) to K(m) (Michaelis-Menten constant) were calculated. For tabun, this value (for the most effective OpdA variant) exceeded any value published to date for other enzymes. The value was within an order of magnitude for the highest value reported for sarin, but there appears to be no published value for ethyl sarin for comparison. The OpdA enzymes were relatively inefficient in degrading soman.

Ricin antitoxins based on lyotropic mesophases containing galactose amphiphiles.

Lyotropic mesophases of the inverse hexagonal or cubic type are nanostructured materials that result from the self-assembly of amphiphilic surfactant molecules in water. The extremely large area of the surfactant-water interface inherent within these structures makes them attractive media for sorbent or encapsulant systems. Here, we report on the development of a new class of polyvalent materials that are based on the incorporation of bioactive ligands within lyotropic mesophases. In particular, we have studied the potential for these materials to behave as polyvalent antitoxins by incorporating synthetic galactose amphiphiles, which mimic the natural cell surface ligand for the protein toxin ricin. The study demonstrates that cubic morphology lyotropic mesophases containing galactose amphiphiles exhibit high specificity ricin uptake, with favorably high dissociation constants and high capacities. We suggest that lyotropic mesophase polyvalent ligands are thus promising materials for the incorporation of a broad range of cell surface recognition moieties and hence may have wide applicability as materials capable of partaking in biological recognition processes.

Monovalent and polyvalent carbohydrate inhibitors of ricin binding to a model of the cell-surface receptor.

A selection of galactose and lactose analogues was evaluated for their potency in inhibiting the binding of ricin to immobilised asialofetuin, which is a model of the cell-surface receptor for ricin. The aim was to identify compounds that could be used as antagonists of ricin toxicity in vivo, and as more selective, and therefore safer, antitoxins. Although one of these analogues had been identified by molecular modelling in a previous study as a potentially potent inhibitor, it and the other carbohydrates studied were less effective than galactose and lactose themselves (I(50) = 1.39 and 0.74 mM, respectively). In an attempt to increase the potency of carbohydrate-based inhibitors, galactose was coupled to the surface of dendrimers. No synergistic interactions were observed from this multivalent approach. Encouraging results, however, were obtained with a self-assembled lyotropic mesophase gel containing novel synthetic galactose-based surfactants, which was able to sequester ricin from aqueous solution in a 2-phase system.

Characterization of the binding of cholera toxin to ganglioside GM1 immobilized onto microtitre plates.

Ganglioside GM1 is the receptor for cholera toxin on cell surfaces, and the binding of cholera toxin to GM1 immobilized on microtitre plates has been reported previously by several authors as an assay for the toxin (GM1-ELISA). This assay has been examined in detail. Results were independent of the adsorption solvent for GM1 (methanol or phosphate-buffered saline), the pH of aqueous solvents (7.4-10.2) and the temperature (4-37 degrees C). High and near-maximal rates of absorbance change in the assay were found for lower concentrations of GM1 (100 ng ml(-1)) and for shorter incubation times (a few hours) than reported in the literature. A method was devised to provide a semi-quantitative estimate of the amount of GM1 bound to the plate; this was found to be in the low nanogram range. Binding of cholera toxin to the immobilized GM1 required > or =1.5 h for maximal assay results. The failure of free GM1 in solution to displace cholera toxin once bound to immobilized GM1 indicated that binding to immobilized GM1 is irreversible in the time frame of the experiment. Data from the literature support the very slow dissociation rates of the toxin-GM1 complex.

Characterization of the Asialofetuin microtitre plate-binding assay for evaluating inhibitors of ricin lectin activity.

Optimum conditions for the binding of ricin to the glycoprotein asialofetuin immobilized on microtitre plates were investigated for the purpose of evaluating inhibitors of ricin B-chain lectin activity. Such inhibitors are of potential value in the use of immunotoxins based on ricin. This assay was first reported in 1986, but has not been characterized fully. Maximum binding of asialofetuin to the plate was observed at a concentration of ca. 4 microg ml(-1). Binding increased with time of incubation (1-24 h), pH (7.4-9.9) and temperature (2-37 degrees C). The pH effects were more marked at lower temperatures. Saturable binding of ricin to immobilized asialofetuin was observed, and at least 80% of maximum binding was observed by 10 min of incubation time. The binding was found to be very tight, such that an appreciable proportion of ricin added to the wells was bound at low concentrations, and binding was only partially reversible by addition of free galactose. Consequently, only estimates of the ricin-asialofetuin and ricin-galactose dissociation constants could be determined: 1.9 nM and 83 microM, respectively. Binding of ricin A- and B-chains was found to be 47% (at a 200-fold higher concentration) and 26% (at a twofold higher concentration) of that of the whole ricin molecule, respectively. The assay permits qualitative comparison of inhibitors of ricin B-chain lectin activity.

The toxicology of microcystins.

Microcystins are a family of more than 50 structurally similar hepatotoxins produced by species of freshwater cyanobacteria, primarily Microcystis aeruginosa. They are monocyclic heptapeptides, characterised by some invariant amino acids, including one of unusual structure which is essential for expression of toxicity. Microcystins are chemically stable, but suffer biodegradation in reservoir waters. The most common member of the family, microcystin-LR (L and R identifying the 2 variable amino acids, in this case leucine and arginine respectively) has an LD50 in mice and rats of 36-122 microg/kg by various routes, including aerosol inhalation. Although human illnesses attributed to microcystins include gastroenteritis and allergic/irritation reactions, the primary target of the toxin is the liver, where disruption of the cytoskeleton, consequent on inhibition of protein phosphatases 1 and 2A, causes massive hepatic haemorrhage. Microcystins are tight-binding inhibitors of these protein phosphatases, with inhibition constants in the nanomolar range or lower. Uptake of microcystins into the liver occurs via a carrier-mediated transport system, and several inhibitors of uptake can antagonise the toxic effects of microcystins. The most effective of these is the antibiotic rifampin (a drug approved for clinical use), which protects mice and rats against microcystin-induced lethality when given prophylactically and, in some cases, therapeutically.

Evaluation of the therapeutic efficacy of some antimuscarinics against soman in vivo.

The therapeutic efficacy of tacrine, atropine and glycopyrrolate alone or in combination with the oxime HI-6 against soman was evaluated in anaesthetized rats. Arterial blood pressure, heart rate, respiratory frequency and body temperature were monitored in vivo. Blood cholinesterases were determined after each drug or soman challenge. At the lowest concentration tested (2.5 mg kg-1), tacrine was effective in improving the survivability of the rat by a factor of 2.6 (protection ratio), whereas the protection by atropine or glycopyrrolate was either insignificant or only marginally effective (protection ratio ranged from 1.0 to 1.9). In combination with HI-6, atropine increased the ratio to 4.6. In contrast, tacrine with HI-6 failed to improve the efficacy of the regimen, while glycopyrrolate plus HI-6 showed only slight improvement. The four physiological parameters monitored were relatively constant during the time course of the experiment in both the control and those with drug therapy. The more noticeable changes occurred toward the end of the experiment when sufficient amount of soman was injected to cause lethality. Death of the animal was usually preceded by a surge of arterial blood pressure and heart rate and a decrease in respiratory frequency. These physiological parameters rapidly deteriorated to zero just before the animal died. Blood and plasma cholinesterases were significantly inhibited after the animal received a relatively small dose of soman (20 micrograms kg-1) and were almost completely inactivated after the lethal dose of soman was administered. However, these changes of enzyme activity did not correspond well with the survivability of the rat. The inclusion of HI-6 with the three antimuscarinics appeared to be capable of protecting some cholinesterases against soman.

Oxime effects on the rate constants of carbamylation and decarbamylation of acetylcholinesterase for pyridostigmine, physostigmine and insecticidal carbamates.

The effects of the oximes 2-pyridine aldoxime methiodide (PAM), HI-6, HS-6, toxogonin and TMB-4 on the rate of carbamylation of membrane-bound bovine erythrocyte acetylcholinesterase were studied. The second-order rate constant of carbamylation (ki) and the first-order rate constant of decarbamylation (k3) were calculated from the proportion of free acetylcholinesterase at equilibrium and the rate of approach to equilibrium. Twenty insecticidal carbamates plus physostigmine and pyridostigmine were studied. The oximes increased ki for several carbamates, with HI-6 causing an increase in the most number of cases (12) and PAM the least (3). HI-6 was also a potent accelerator of decarbamylation (increase in k3) in all cases, whereas PAM caused a significant decrease in k3 in 15 cases and a nonsignificant decrease in the other 7. Toxogonin and TMB-4 increased k3 or had no significant effect. The results were generally consistent with a proposal in the literature that there is a correlation between increased ki and increased toxicity of the carbamate in the presence of an oxime.

Review of oximes available for treatment of nerve agent poisoning.

A review was conducted of papers describing the use of N-methyl-2-pyridinealdoxime (PAM), toxogonin or HI-6 as antidotes to the nerve agents tabun, sarin, soman and VX. The review included use of the oxime alone, oxime plus atropine and oxime plus atropine plus diazepam, given therapeutically, i.e. after nerve agent, in all cases. Experiments with any of these compounds given prophylactically were not considered. The review also included protocols of pyridostigmine prophylaxis and oxime-atropine therapy (with or without diazepam). It was difficult to draw conclusions as to the best oxime to use, because of lack of data in many cases. The identity of the oxime did not appear to be important when pyridostigmine prophylaxis was combined with atropine-oxime-diazepam therapy; in these cases, very good protection was observed in guinea pigs against all four nerve agents. The choice of oxime based on the data presently available may well depend on factors other than protection against lethality, such as cost and availability of the oxime and human toxicity of the oxime. This last factor was also reviewed, and the results showed that toxogonin is likely to cause more side-effects than PAM or HI-6. The efficacy of the oximes against the emerging threat agent GF was also reviewed.

Rate constants of carbamylation and decarbamylation of acetylcholinesterase for physostigmine and carbaryl in the presence of an oxime.

Membrane-bound bovine erythrocyte acetylcholinesterase was inhibited with physostigmine or carbaryl, and the rate constants of carbamylation and decarbamylation were determined from the proportion of inhibited acetylcholinesterase in the steady state, and the rate of approach to the steady state. The oximes 2-PAM, HI-6, HS-6, TMB-4 and toxogonin, at 0.1 mM, all decreased the rate of carbamylation by physostigmine, but increased the rate of carbamylation by carbaryl. TMB-4 and toxogonin were the most effective oximes in potentiating carbamylation by carbaryl, with an enhancement of the second-order rate constant of 54- and 17-fold respectively. The greatest reduction in the rate constant for carbamylation by physostigmine (3.7-fold) was caused by HI-6. HS-6 and HI-6 increased the rate of decarbamylation, while 2-PAM reduced the rate of decarbamylation if physostigmine was the carbamate. 2-PAM and HI-6 were also studied with soluble bovine erythrocyte acetylcholinesterase, and similar results were obtained. The results extend those in a recent report by other authors who studied the half-life of carbamylation for acetylcholinesterase and butyrylcholinesterase in an attempt to understand the mechanism by which oximes increase the toxicity of carbaryl in vivo. These authors proposed binding of the oximes to an allosteric site on the enzyme. While not discounting this possibility, the present results, taken with other reports in the literature, suggest that binding of the oximes to the anionic subsite of the active site of the enzyme is also feasible. The present results also offer an explanation for another recent report, in which anomalous results were presented for decarbamylation of physostigmine-inhibited and carbaryl-inhibited erythrocyte acetylcholinesterase in the presence of 2-PAM or HI-6.

The diacetylmonoxime assay of urea, its application to the assay of diacetylmonoxime and a comparison with other methods for the analysis of diacetylmonoxime.

The experiments reported herein were designed to identify the best assay of diacetylmonoxime (DAM) in biological fluids, with particular emphasis on detection limits. Initially, four variations of the assay of urea with excess DAM were compared. The best method, published in 1977, was one that includes thiosemicarbazide, 4-aminoantipyrine and ceric ammonium sulphate in the acid reagent; it is fast, gives a reasonably stable chromophore and displays good linearity. However, the reaction was two or more times less sensitive when applied to the assay of DAM, with urea in excess, by interchanging the amounts of urea and DAM. Further, the calibration graphs did not pass through the origin, and one of the methods gave a mixture of two chromophores. None approached the sensitivity of a DAM-urea reaction specifically designed to assay biacetyl (formed from DAM by acid hydrolysis) and published in 1968. This method, using antipyrine and arsenicosulphuric acid, is also fast, with good linearity and a stable chromophore, but is sensitive to interference by plasma and urine. An alternative photometric assay that does not involve urea was equally sensitive. It had the advantage of less interference by plasma and urine but was more time-consuming. Both of these photometric methods had a limit of detection of ca. 0.2 microgram DAM, similar to that of a high-performance liquid chromatography (HPLC) assay. Sample clean-up is necessary before application of the HPLC assay.

Effects of the nerve agents soman and tabun on the uptake and release of GABA and glutamate in synaptosomes of guinea pig cerebral cortex.

1. Crude and purified synaptosomes were prepared from the cerebral cortex of the rat or the guinea pig and used to study the uptake and release of [3H]GABA and [3H]glutamate. 2. Baclofen at 10(-5) M inhibited stimulated release of [3H]GABA from crude rat and guinea pig synaptosomes, but not from purified rat synaptosomes. 3. 1-2 mM tabun decreased the uptake of [3H]GABA and increased the uptake of [3H]glutamate by purified guinea pig synaptosomes. 4. Soman and tabun at 10(-6) M and 10(-5) M inhibited basal release of [3H]GABA and [3H]glutamate from crude guinea pig synaptosomes. Tabun at 10(-5) M decreased stimulated release of [3H]GABA while soman had no effect. 5. The results do not sustain the possibility that nerve agents cause convulsions by affecting the uptake or release of GABA or glutamate. However indirect evidence was obtained that soman and tabun inhibit catabolism of GABA and glutamate.

Assessment of a model for the opposing effects of N-ethylmaleimide on the affinity of muscarinic agonists for M2 receptors.

1. Homogenates of guinea pig right atrium (M2 receptors) were treated with N-ethylmaleimide, after which the ability of carbachol to inhibit binding of [3H]quinuclidinyl benzilate was studied. 2. At 37 degrees C, 10(-4) M and 10(-3) M, but not 10(-5) M, N-ethylmaleimide increased the affinity of carbachol for the receptor. At 2 degrees C, 10(-5) M and 10(-4) M, but not 10(-3) M, N-ethylmaleimide decreased carbachol affinity. At 2 degrees C, after the homogenate had been incubated at 37 degrees C without N-ethylmaleimide, 10(-5) M N-ethylmaleimide decreased carbachol affinity, as at 2 degrees C without preincubation, but 10(-3) M N-ethylmaleimide increased carbachol affinity, as at 37 degrees C. 10(-4) M N-ethylmaleimide was without effect. 3. The results are discussed with respect to a previously proposed model in which N-ethylmaleimide interacts with two sites, causing an increase or decrease in agonist affinity respectively.

The interaction of tacrine with benzodiazepine and GABA binding sites of guinea pig brain.

Tacrine (1,2,3,4-tetrahydro-9-acridinamine) inhibited binding of [3H]flunitrazepam to benzodiazepine receptors of guinea pig hippocampus with an inhibition constant of 46 microM at 2 degrees C and 37 degrees C. gamma-Aminobutyric acid (GABA) decreased the affinity of tacrine for the receptor, suggesting that tacrine may act as an inverse agonist. A Hill coefficient less than 1 was observed under all conditions. Allosteric interactions may explain this behaviour, since 100 microM tacrine increased the rate of dissociation of [3H]flunitrazepam from the receptor. Tacrine inhibited the binding of 11 nM [3H]GABA to GABA receptors of guinea pig cerebral cortex with I50 = 188 microM. Bicuculline methiodide was 4 times as potent (I50 = 49 microM). The interaction of tacrine with GABA or benzodiazepine binding sites is unlikely to be of clinical significance.

Reversibility of the inhibition of acetylcholinesterase by tacrine.

Inhibition of bovine erythrocyte acetylcholinesterase (AChE) by 1,2,3,4-tetrahydro-9-acridinamine (tacrine) was independent of time of incubation and was partially reversed by dilution and by increased substrate concentration. It was fully reversed by dialysis. Similar results were obtained with AChE from other sources. The results are consistent with some reports in the literature, but not with others; none of these reports examined all four criteria of reversibility. The results do not explain the prolonged inhibition of AChE in vivo or the ability of tacrine to protect animals against the lethal effects of organophosphate anticholinesterases.

Opposing effects of N-ethylmaleimide on the affinity of carbachol for muscarinic cholinoceptors of guinea-pig atrium.

1. Inhibition of the binding of [3H]quinuclidinyl benzilate to homogenates of guinea pig right atrium (M2 receptors) by varying concentrations of carbachol was studied. 2. Pretreatment of membranes with 5 x 10(-5) M N-ethylmaleimide at 2 degrees C shifted the carbachol inhibition curve to the right, indicating decreased affinity of the receptor for carbachol. However pretreatment at 37 degrees C moved the curve to the left. 3. The ability of guanyl-5'-yl imidodiphosphate to reduce agonist affinity was largely eliminated by treatment with N-ethylmaleimide at both temperatures. 4. Conflicting reports in the literature and the present results can be explained by invoking a model in which N-ethylmaleimide has a high affinity for a heat-labile site and a lower affinity for a heat-insensitive site. Reaction with the first site decreases agonist affinity, but at 37 degrees C this site is largely inactivated and reaction with the second site, which leads to increased agonist affinity, predominates.

Factors influencing the calculation of results from studies of the release of tritiated neurotransmitters from superfused slices of guinea pig striata.

Slices of guinea pig striata were incubated with tritiated choline, dopamine, or serotonin and the release of radioactive transmitter was studied in a superfusion system. Some experiments were also done on the release of [3H]acetylcholine from rat striatal slices. For analysis of the results, various parameters of the system were determined in order to establish the most reliable method of assessing drug effects on transmitter release. Peaks of radioactivity (S1 and S2) above basal release of radioactivity (B1 and B2) were observed after two depolarizations of the nerve endings with high K+ buffer. Each stimulation was for 2 min, and S2 occurred 20 min after S1. There was a highly significant correlation between S1 and the protein content of the slices for acetylcholine release from guinea pig striata. Basal release of radioactivity, and the ratio S2/S1, were not sensitive to minor changes in the experimental conditions. It was concluded that S2/S1, rather than S1 or S1/B1, should be used as a measure of drug effects on release. The experiments also demonstrated that re-uptake of released neurotransmitter is operative in the superfusion system for dopamine, but not for serotonin. Differences were observed between the rat and the guinea pig with respect to the release of [3H]acetylcholine.

Tacrine slows the rate of ageing of sarin-inhibited acetylcholinesterase.

Bovine erythrocyte acetylcholinesterase was inhibited by the organophosphate sarin, and the rate of ageing (the time-dependent decrease in the ability of an oxime to reactivate the enzyme) was studied. At pH 7.0 and 37 degrees C, 10(-5) M or 10(-6) M tacrine (tetrahydroaminoacridine) decreased the rate of ageing in low ionic strength buffer. Tacrine at 10(-5) M also significantly decreased the rate of ageing in 150 mM NaCl. The results indirectly demonstrated that the inhibition of substrate hydrolysis by tacrine is reversible, and that tacrine does not prevent reactivation of sarin-inhibited acetylcholinesterase. Both these observations, which were also made for rat brain acetylcholinesterase, are in contrast with reports in the literature.