Cyanide intoxication: protection with oxygen.

Prophylactic protection against cyanide intoxication in mice can be enhanced by the administration of oxygen, especially when it is used in combination with the conventional cyanide antidotes, sodium nitrite and sodium thiosulfate. The LD(50) values were compared in groups of mice premedicated with sodium thiosulfate or sodium nitrite, or both, in air and in oxygen. These results indicate that oxygen alone provides only minimal protection. Although oxygen enhances the protective effect of sodium thiosulfate to a minor degree, it does not enhance the protection of sodium nitrite at all; and yet, it potentiates the effectiveness of a combination of these two antagonists against cyanide intoxication.

Comparing therapeutic and prophylactic protection against the lethal effect of paraoxon.

Prophylactic and therapeutic efficacy against organophosphorus (OP) intoxication by pralidoxime (2-PAM) and atropine were studied and compared with sterically stabilized long-circulating liposomes encapsulating recombinant organophosphorus hydrolase (OPH), either alone or in various specific combinations, in paraoxon poisoning. Prophylactic and therapeutic properties of atropine and 2-PAM are diminished when they are used alone. However, their prophylactic effects are enhanced when they are used in combination. Present studies indicate that sterically stabilized liposomes (SL) encapsulating recombinant OPH (SL-OPH) alone can provide much better therapeutic and prophylactic protection than the classic 2-PAM + atropine combination. This protection was even more dramatic when SL-OPH was employed in combination with 2-PAM and/or atropine: the magnitude of prophylactic antidotal protection was an astounding 1022 LD(50) [920 mg/kg (LD(50) of paraoxon with antagonists)/ 0.95 mg/kg (LD(50) of control paraoxon)], and the therapeutic antidotal protection was 156 LD(50) [140 mg/kg (LD(50) of paraoxon with antagonists)/0.9 mg/kg (LD(50) of control paraoxon)]. The current study firmly establishes the value of using liposome encapsulating OPH.

Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.

These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.

Antagonism of the lethal effects of cyanide with resealed erythrocytes containing rhodanese and thiosulfate.

A new concept has been presented for the antagonism of cyanide and possibly other chemical toxicants. Until now, only a half dozen truly specific "antidotes" were known. There are many other "antidotes" which merely prevent the absorption or enhance the elimination of a toxic compound rather than specifically destroying the substance to prevent its toxic effect. This new approach has considerable conceptual significance in toxicology, as it suggests the encapsulating other enzymes to degrade various other chemical toxicants. There are many chemical toxicants for which there are no specific antidotes, and the conceptual approach of employing erythrocyte-encapsulated enzyme provides an innovative, specific approach to antagonize the toxic and lethal effects of these chemicals.

Fluorometric determination of cyanide in biological fluids and pyridoxal.

Investigations of the physiological disposition of cyanide during thiosulfate therapy has necessitated the development of a convenient and sensitive method for cyanide analysis. A fluorometric method involving the catalytic conversion of pyridoxal to 4-pyridoxylactone was adapted for use with biological fluids by employing microdiffusion analysis. The presence of the cyanide antagonist, sodium thiosulfate, interferes with the formation of the fluorophore. In order to circumvent this interference, the pH of the diffusion media was altered to selectively diffuse cyanide. After testing various acidifying agents, an acetate buffer (pH = 5.2) was determined to be satisfactory. The fluorometric method was then correlated with the classical colorimetric procedure by an in vivo study. Blood from mice treated with sodium nitrite and sodium thiosulfate, prior to receiving potassium cyanide, was analyzed by both procedures and no significant difference was demonstrated between the results of the two methods of analysis.

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism.

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.

Cyanide intoxication in sheep: enhancement of efficacy of sodium nitrite, sodium thiosulfate, and cobaltous chloride.

For treatment of cyanide intoxication of ruminants, the present recommended doses of sodium nitrite (5 mg/kg of body weight) and sodium thiosulfate (25 to 50 mg/kg) are smaller than those recommended for other animals; the decrease is partially attributed to the greater susceptibility of ruminants to the toxic effects of sodium nitrite. Based on the high tissue concentration and activity rate of rhodanese in ruminants, sulfur donors such as sodium thiosulfate could be utilized more efficaciously. Doses of sodium nitrite and sodium thiosulfate (up to 22 and 660 mg/kg, respectively) were evaluated in the present studies. Adjustment of the antidotal combination provided almost three times the protection afforded by the previously recommended doses. Moreover, under the conditions tested, the newly adjusted dose levels of sodium thiosulfate alone were more effective than the previously used antidotal combination of sodium nitrite and sodium thiosulfate and this protective effect was enhanced by cobaltous chloride (10.6 mg/kg) or sodium nitrite. The present recommended therapeutic approach to cyanide intoxication in sheep should be based primarily on administration of a much higher dose of sodium thiosulfate in combination with sodium nitrite or cobaltous chloride (or both).

Cyanide intoxication in sheep: therapeutic value of oxygen or cobalt.

The combination of cobalt salts and oxygen with the traditional sodium nitrite-sodium thiosulfate antidote may have value as cyanide antidotes. Results reported previously in mice were extended to sheep in the present experiments. Cobaltous chloride (15 mg/kg of body weight) or oxygen with or without sodium nitrite and sodium thiosulfate were compared with respect to the median lethal dose of oral sodium cyanide. Although cobaltous chloride or oxygen used alone did result in a significant increase in the median lethal dose of sodium cyanide in sheep, the protection was minor compared with the overall protection provided by the classic antidotal combination of sodium nitrite and sodium thiosulfate. Cobaltous chloride combined with sodium nitrite and sodium thiosulfate did not provide any increase in protection more than that observed with sodium nitrite and sodium thiosulfate alone. Oxygen used in combination with sodium nitrite and sodium thiosulfate did significantly increase the protection. However, the increase in protection was not of sufficient magnitude to recommend it as a practicable addition to the present therapeutic regimen.

Effects of oxygen on the antagonism of cyanide intoxication: cytochrome oxidase, in vitro.

Since oxygen was reported to be an effective cyanide antagonist in vivo, particularly in the presence of the classic antidotal combination of sodium nitrite and sodium thiosulfate, in vitro studies were initiated in an attempt to investigate the mechanism of oxygen-mediated cyanide antagonism. The effect of oxygen on cyanide-inhibited cytochrome oxidase with and without cyanide antagonist(s) was investigated in a purified membraneous enzyme system prepared from rat liver mitochondria. Cyanide produced a concentration dependent inhibition of cytochrome oxidase, and 100% oxygen did not alter the inhibition produced by KCN either in the presence or absence of sodium thiosulfate. However, the addition of sodium thiosulfate and rhodanese to the assay reactivated the cyanide-inhibited cytochrome oxidase. Kinetic analysis indicated rhodanese competes with cytochrome oxidase for cyanide, and oxygen had no effect on this coupled reaction. In conclusion, the in vivo antidotal properties of oxygen cannot be attributed to oxygen-mediated reactivation of cyanide-inhibited cytochrome oxidase or an oxygen-mediated acceleration of rhodanese detoxification.

Antagonism of the lethal effects of paraoxon by carrier erythrocytes containing phosphotriesterase.

Annealed murine erythrocytes were employed as a carrier model to antagonize the toxic effects of organophosphorus agents. These resealed cells containing a recombinant phosphotriesterase provided striking protection against the lethal effect of paraoxon, an active metabolite of an agricultural pesticide, parathion. Phosphotriesterase hydrolyzes paraoxon to the less-toxic 4-nitrophenol and diethylphosphate. This enzyme was encapsulated into carrier erythrocytes by hypotonic dialysis with subsequent resealing and annealing. These carrier cells were administered to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recipient animals were subsequently challenged with paraoxon and a marked protection was noted. Protection of free enzyme and encapsulated enzyme was compared and the encapsulated enzyme was found to persist longer and possess much greater efficacy. Less serum cholinesterase inhibition also was observed with this enhanced protection. These results indicate that the erythrocyte carrier alone is quite effective in the antagonism of organophosphorus intoxication. Moreover, when these carrier cells were administered in combination with 2-PAM and/or atropine, a marked synergism was observed.

Bond strength of light-cured glass ionomers to carious primary dentin.

The pre-cooperative or handicapped child with decay presents a special challenge to the practitioner and may require sedation or general anesthesia. Treatment with an interim restoration may delay treatment until the child is more mature and can accept dental treatment and is a more conservative approach than sedation, extractions or general anesthesia. Glass ionomer materials have been utilized for this application, but little is known about their retention to carious dentin. The purpose of this study was to determine whether the presence of artificial dentin decay will affect the shear bond strength of two light-cured glass ionomer materials. VariGlass and Vitrebond glass ionomer materials were attached to carious and non-carious primary dentin surfaces and bond strengths determined. There were no significant differences in shear bond strengths between the decayed and non-decayed surfaces [p < or = .001]. VariGlass had higher shear bond strengths than Vitrebond only after a pre-treatment with the PAA containing liquid. Pre-treatment with the liquid provided with each light-cured glass ionomer was beneficial in all instances except for Vitrebond on non-decayed surfaces.

Encapsulation of thiosulfate: cyanide sulfurtransferase by mouse erythrocytes.

Murine carrier erythrocytes, prepared by hypotonic dialysis, were employed in the encapsulation of several compounds including [14C]sucrose, [3H]inulin, and bovine thiosulfate:cyanide sulfurtransferase (rhodanese), a mitochondrial enzyme which converts cyanide to thiocyanate. Approximately 30% of the added [14C]sucrose, [3H]inulin, and rhodanese was encapsulated by predialyzed erythrocytes, and a decrease in the mean corpuscular volume and mean corpuscular hemoglobin was observed. In the encapsulation of rhodanese a recovery of 95% of the erythrocytes was achieved and an 85% equilibrium was established. The addition of potassium cyanide (50 mM) to intact, rhodanese-loaded erythrocytes containing sodium thiosulfate resulted in its metabolism to thiocyanate. These results establish the potential use of erythrocytes as biodegradable drug carrier in drug antagonism.

Recent perspectives on the toxicodynamic basis of cyanide antagonism.

The mechanism of action of nitrite-thiosulfate (Chen et al., 1933a ,b; Hug , 1933) in the antagonism of the lethal effects of cyanide is much more complex than proposed 50 years ago. Some of the recent findings concerning the mechanism of nitrite action have conceptual theoretical and practical significance, as the development of newer cyanide antagonists are dependent on the elucidation of the basic mechanism of antidotal action. There are preliminary evidence which suggest a vasogenic action rather than methemoglobin formation is the primary action of nitrite, as a cyanide antagonist. Various vasogenic compounds have been uncovered and they may play an important role in the future development of a new class of cyanide antagonists. Also recent development in thiol detoxication of cyanide suggest that rhodanese may play a more complex role. The detoxification of cyanide may be viewed from a considerably more complex perspective with the elucidation of recent mechanisms. It also may provide a newer conceptual basis for a more rational development of future compounds to antagonize the lethal effects of cyanide.

A nuclear magnetic resonance study of nicotinamide adenine dinucleotide phosphate binding to Lactobacillus casei dihydrofolate reductase.

The binding of NADP+ to dihydrofolate reductase (EC 1.5.1.3) in the presence and absence of substrate analogs has been studied using 1H and 13C nuclear magnetic resonance (NMR). NADP+ binds strongly to the enzyme alone and in the presence of folate, aminopterin, and methotrexate with a stoichiometry of 1 mol of NADP+/mol of enzyme. In the 13C spectra of the binary and ternary complexes, separate signals were observed for the carboxamide carbon of free and bound [13CO]NADP+ (enriched 90% in 13C). The 13C signal of the NADP+-reductase complex is much broader than that in the ternary complex with methotrexate because of exchange line broadening on the binary complex signal. From the difference in line widths (17.5 +/- 3.0 Hz) an estimate of the dissociation rate constant of the binary complex has been obtained (55 +/- 10 sec-1). The dissociation rate of the NADP+-reductase complex is not the rate-limiting step in the overall reaction. In the various complexes studied large 13C chemical shifts were measured for bound [13CO]NADP+ relative to free NADP+ (upfield shifts of 1.6-4.3 ppm). The most likely origin of the bound shifts lies in the effects on the shieldings of electric fields from nearby charged groups. For the NADP+-reductase-folate system two 13C signals from bound NADP+ are observed indicating the presence of more than one form of the ternary complex. The IH spectra of the binary and ternary complexes confirm both the stoichiometry and the value of the dissociation rate constant obtained from the 13C experiments. Substantial changes in the IH spectrum of the protein were observed in the different complexes and these are distinct from those seen in the presence of NADPH.

Effect of antagonists on the physiologic disposition of sodium cyanide.

Attempts were made to evaluate the effects of pretreatment with air and oxygen either alone or in various combinations with sodium nitrite and/or sodium thiosulfate on the physiological disposition of 14C-labeled sodium cyanide in mice. The radioactive respiratory excretion was studied by radiorespirometry, and the effects of various combinations of cyanide antagonists were compared. Oxygen either alone or in combination with sodium thiosulfate significantly enhanced the respiratory excretion when compared with air. Sodium thiosulfate accelerated the initial rate, but not the total amount of radioactivity excreted. The cumulative recovery of radioactive gases was significantly greater with groups receiving oxygen either alone or with sodium thiosulfate. When sodium nitrite was employed as an antidote either alone or with sodium thiosulfate, no difference in the respiratory excretion was noted between air and oxygen. The use of the sodium nitrite-sodium thiosulfate combination either with air or oxygen resulted in a marked decrease in the initial rate as well as the total amount of respiratory radioactivity excreted. No significant differences between various experimental groups were noted in the total amount of urinary radioactivity excreted or the total body retention of radioactivity.

Encapsulation of rhodanese and organic thiosulfonates by mouse erythrocytes.

A series of organic thiosulfonates were synthesized and studied as sulfur donor substrates for rhodanese encapsulated within murine carrier erythrocytes. Previous studies have indicated that resealed erythrocytes containing rhodanese (CRBC) and sodium thiosulfate can rapidly metabolize cyanide to the less toxic thiocyanate. This thiosulfate-rhodanese system was very efficacious as a new conceptual approach to antagonize cyanide intoxication both in vitro and in vivo. However, its potential is restricted because of the limited availability of thiosulfate due to its poor permeability through RBC membrane. Present studies suggest that there are advantages in using alternative sulfur donors, i.e., organic thiosulfonates in this rhodanese-containing resealed erythrocyte system, since these compounds have higher lipid solubility than inorganic thiosulfates and can readily penetrate the red blood cell membrane. Therefore, this system could provide a virtually unlimited amount of sulfur donor to the encapsulated rhodanese even if the substrates are in solution outside the cells. Moreover, the rhodanese reaction rate of any of these organic thiosulfonates is much faster than the rate observed with the classic cyanide antidote, sodium thiosulfate. This CRBC system will continue to detoxify cyanide even when these encapsulated sulfur donors are depleted, as the lipid soluble organic thiosulfonate outside the cells will diffuse past the membrane into the cell to replenish the sulfur donor. The encapsulation efficiency for rhodanese is about 30%, and the velocity of the rhodanese reaction increases linearly with the volume of enzyme-laden erythrocytes. Similarly, reaction velocity increases linearly with substrate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)