Characterization of liposomal vesicles encapsulating rhodanese for cyanide antagonism.

The major mechanism of removing cyanide from the body is its enzymatic conversion by a sulfurtransferase, e.g. rhodanese, to the less toxic thiocyanate in the presence of a sulfur donor. Earlier results demonstrated that externally administered encapsulated rhodanese significantly enhances the in vivo efficacy of the given sulfur donor. Present studies are focused on liposomal carrier systems encapsulating rhodanese. Physicochemical properties, e.g. membrane rigidity, size distribution, surface potential, osmolarity, and viscosity, were determined for various liposomal lipid compositions and hydrating buffers to establish in vitro stability and in vivo fate. Lipid composition was also optimized to achieve maximum encapsulation efficiency.

Anionic currents in hypoxia-mediated cardiac toxicity: a computer study.

Hypoxia-caused modulation of cardiac electrophysiology was modeled by computer simulation. Emphasis was on the effect of activation of anionic channels on the electrical state of the tissue. The model includes implicitly the effect of the presence of reactive oxygen species (ROS) and nitrogen oxide (NO) on myocyte membrane voltage by their contribution to the activation of chloride currents. Three anionic currents were added to the modified Luo-Rudy ionic model of the ventricular action potential used in these calculations. The effect of the activation of the usually dormant currents due to hypoxia results in the modulation of the morphology of the action potential and the ECG. Transition of the ECG to ventricular fibrillation is shown. An important finding reported here is that control of the swelling and protein kinase C (PKC)-activated chloride currents can limit the electrical chaos of pharmacologically-caused hypoxic cardiac toxicity.

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.

Toxicology update: the cardiotoxicity of the oxidative stress metabolites of catecholamines (aminochromes).

This toxicology update reviews the oxidative stress metabolites of catecholamines, postulated to be the biochemical initiators of cardiotoxicity. A brief overview of catecholamine metabolism is provided with several noteworthy historical observations relating to the autoxidation and rearrangement of epinephrine. The basic chemical and physical properties of adrenochrome and adrenolutin are discussed. The autoxidative, enzymatic and cellular basis for the transformation of catecholamines to oxidative metabolites is reviewed. Mechanisms seeking to account for the observed cardiotoxic changes in isolated heart perfusion studies and in vivo models are described.

In vitro effects of anionic sulfur compounds on the spectrophotometric properties of native DNA.

Several anionic sulfur compounds are recognized as efficacious pretreatments for sulfur mustard (HD) poisoning. Our intent was to see if pretreatment compounds had a direct effect on DNA, a site where HD damage is thought to occur. A modification of the method of Szinicz et al. (Arzneim.-Forsch. 1981; 31: 1,713-1,717) was used to analyze the UV/VIS spectrum (205-400 nm) (n = 6) of calf thymus DNA (10-15 x 10(3) kDa) in the absence or presence of increasing concentrations of sodium thiosulfate, sodium 2-aminoethanethiosulfonate (thiotaurine), sodium metabisulfite or sodium sulfate. All compounds produced concentration-dependent absorbance decreases primarily at 212 nm, but also at 259 nm, with the exception of sodium sulfate. For example, 8.36 x 10(-4) M sodium thiosulfate reduced the absorbance of DNA at 212 nm by >60%. The kinetics of sulfur compounds on native DNA need further study. We propose that these anionic sulfur compounds interact with DNA possibly by changing the topology of this macromolecule. Effects may be due to interactions of these sulfur compounds at higher concentrations with DNA, with resulting ligand-DNA supercoiling. This process could protect against HD intoxication, which is caused in part by the uncoiling of DNA.

In vitro and in vivo comparison of sulfur donors as antidotes to acute cyanide intoxication.

Antidotes for cyanide (CN) intoxication include the use of sulfane sulfur donors (SSDs), such as thiosulfate, which increase the conversion of CN to thiocyanate by the enzyme rhodanese. To develop pretreatments that might be useful against CN, SSDs with greater lipophilicity than thiosulfate were synthesized and assessed. The ability of SSDs to protect mice against 2LD50 of sodium cyanide (NaCN) administered either 15 or 60 min following administration of an SSD was assessed. To study the mechanism of action of the SSD, the candidate compounds were examined in vitro for their effect on rhodanese and 3-mercaptopyruvate sulfurtransferase (MST) activity under increasing SSD concentrations. Tests were conducted on nine candidate SSDs: ICD1021 (3-hydroxypyridin-2-yl N-[(N-methyl-3-aminopropyl)]-2-aminoethyl disulfide dihydrochloride), ICD1022, (3-hydroxypyridin-2-yl N-[(N-methyl-3-aminopropyl)]-2-aminoethyl disulfide trihydrochloride), ICD1584 (diethyl tetrasulfide), ICD1585 (diallyl tetrasulfide), ICD1587 (diisopropyl tetrasulfide); ICD1738 (N-(3-aminopropyl)-2-aminoethyl 2-oxopropyl disulfide dihydrochloride), ICD1816 (3,3'-tetrathiobis-N-acctyl-L-alanine), ICD2214 (2-aminoethyl 4-methoxyphenyl disulfide hydrochloride) and ICD2467 (bis(4-methoxyphenyl) disulfide). These tests demonstrated that altering the chemical substituent of the longer chain sulfide modified the ability of the candidate SSD to protect against CN toxicity. At least two of the SSDs at selected doses provided 100% protection against 2LD50 of NaCN, normally an LD99. All compounds were evaluated using locomotor activity as a measure of potential adverse behavioral effects. Positive hypoactivity relationships were found with several disulfides but none was found with ICD1584, a tetrasulfide. Separate studies suggest that the chemical reaction of potassium cyanide (KCN) and cystine forms the toxic metabolite 2-iminothiazolidine-4-carboxylic acid. An alternative detoxification pathway, one not primarily involving the sulfur transferases. may be important in pretreatment for CN intoxication. Although studies to elucidate the precise mechanisms are needed. it is clear that these newly synthesized compounds provide a new rationale for anti-CN drugs, with fewer side-effects than the methemoglobin formers.

Comparison of hematologic consequences and efficacy of p-aminophenones in mice.

Controlled methemoglobin (MHb) formation is one strategy employed to counter cyanide (CN) toxicity. Currently available MHb formers present certain drawbacks and limitations. The purpose of this study was to characterize, in mice, the hematologic effects of the MHb-forming compound p-aminopropiophenone (PAPP), and two structurally-related p-aminophenones, p-aminoheptanoylphenone (PAHP) and p-aminooctanoylphenone (PAOP). Although these three p-aminophenones have been shown previously to be efficacious as pretreatments against CN, a more complete understanding of their hematologic effects is lacking. In addition, because the active form of PAPP has been shown to be its N-hydroxy metabolite, the N-hydroxy metabolites of PAPP, PAHP and PAOP were also tested. Using a hemoximeter, blood samples obtained -2 to +180 min relative to intramuscular (i.m.) or intraperitoneal (i.p.) drug injections were evaluated. Sodium nitrite (NaNO(2)) and the appropriate solvents served as the positive and negative controls, respectively. Dose-, time-, route-, and compound-related effects were observed. MHb and sulfhemoglobin levels increased, whereas levels of those parameters related to oxygen-carrying capacity of the blood, such as, oxygen saturation and oxyhemoglobin decreased. In general, the effects of PAHP and PAOP were longer lasting than those of PAPP and NaNO(2). Furthermore, PAPP and NaNO(2) were equally effective with either route of administration. Conversely, PAHP and PAOP showed larger effects when administered i.p. versus i.m. The animals treated with N-hydroxy metabolites of the p-aminophenones also showed similar changes in the hematological parameters measured. N-hydroxy PAPP was shown to be the most rapidly acting MHb-forming compound examined in this series. It could achieve therapeutic concentrations of MHb within 2 min and thus may be considered as a treatment for CN intoxication. Although additional work is needed, these data provide information that will be useful for the successful development of improved anti-CN MHb formers.

In vivo detoxification of cyanide by cystathionase gamma-lyase.

The results of several in vitro studies have suggested that the enzyme cystathionase gamma-lyase (EC 4.4.1.1) may function in the endogenous detoxification of cyanide; however, this possibility has not been investigated in vivo. If cystathionase gamma-lyase in involved in the endogenous detoxification of cyanide, it logically follows that inhibiting cystathionase gamma-lyase should increase the toxicity of cyanide. To test this hypothesis, the activity of cystathionase gamma-lyase was inhibited with a suicide inhibitor, 2-amino-4-pentynoic acid (propargyl-glycine). The activity of liver cystathionase gamma-lyase activity was decreased 96.8% by administration of propargylglycine, indicating that the propargylglycine treatment was effective. The propargylglycine treatment did not alter the activity of thiosulfate:cyanide sulfurtransferase (EC 2.8.1.1) or 3-mercaptopyruvate:cyanide sulfurtransferase (EC 2.8.1.2), two other enzymes that have been proposed to be involved in the detoxification of cyanide. The LD50 of cyanide in rats treated with propargylglycine was 5.14 +/- 0.029 mg NaCN/kg, which was significantly (P < 0.05) lower than the 5.98 +/- 0.008 mg NaCN/kg LD50 of cyanide determined in control rats. The results of these studies suggest that cystathionase gamma-lyase may participate in the detoxification of cyanide in vivo.

The effects of EDRF/NO releasers or calcium ionophore A23187 on cyanide toxicity in mice.

Cyanide (CN) is a well-recognized poison whose complete actions are unclear. It has been shown that a vasoactive role may be partially responsible for the toxic effects of CN. Sodium nitrite, a known methemoglobin former and vasodilator, has been used to treat CN toxicity. It is rapidly transformed to nitric oxide (NO) which is thought to be endothelium-derived relaxing factor (EDRF). Since the literature suggests that NO can influence the biological effects of CN, studies were undertaken to determine if compounds known to release EDRF/NO will modify CN toxicity. Mice were administered a series of compounds which act through EDRF/NO release. These substances included, platelet-activating factor (PAF), hydralazine, bradykinin, histamine, calcium ionophore A23187, carbachol, or substance P at 0.060, 98.7, 50.0, 125, 1.0, 2.26, and 1.0 mg/kg, respectively. As a control, NG-monomethyl-L-arginine (NMA) 70 mg/kg, which inhibits NO synthesis, was administered to mice iv (tail vein) in combination with each test compound. All test compounds and NMA were administered prior to NaCN: NMA, 5 min; carbachol, 0.5 min; hydralazine, 0.5 min; bradykinin, 1 min; histamine, 1 min; substance P, 4 min; PAF, 5 min; and A23187, 5 min. Dose-response relationships were analyzed by probit dose-response methods and protective ratios for each compound were computed. Results suggest (i) that a portion of the action of CN is affected by a particular EDRF/NO-releasing compound, suggesting that each drug specifically affects regional EDRF/NO receptor sites, and (ii) that NO can play a role as a component in CN intoxication. It is suggested that CN does not act uniformly on all EDRF/NO receptor sites to produce toxicity and site-specific EDRF/NO agents may be useful for treating CN.

Temperature effects in cyanolysis using elemental sulfur.

As part of our studies directed at new treatments for cyanide poisoning we examined the effect of temperature on both the non-catalyzed and the albumin-catalyzed reactions of cyanide with a colloidal suspension of elemental sulfur (CSES). Using saturated sulfur solutions prepared in two solvents, pyridine (PY) and methyl cellosolve (MC), the reactions were studied at 15.0, 25.0, 30.0 and 37.5 degrees C. For all the cyanolysis reactions (non-catalyzed and albumin-catalyzed) there is an enhancement of reaction rate when the organic solvent for the sulfur is MC. Irrespective of the solvent for the CSES, the non-catalyzed reactions gave linear Arrhenius plots (PY, correlation coefficient = 0.998; MC, correlation coefficient = 0.997). In each case the entropy of activation was positive (14.1 cal K-1 mol-1 for PY and 56.4 cal K-1 mol-1 for MC). In contrast with these results the albumin-catalyzed reactions generated non-linear Arrhenius plots and negative entropies of activation. Non-linear plots were observed with the three albumins studied: human serum albumin, heat-shock bovine serum albumin and fatty acid-free bovine serum albumin. The non-linear plots are the result of a more complex reaction sequence than a simple cyanolysis reaction.

Cyanide toxicity in mice pretreated with diethylamine nitric oxide complex.

1. Since the literature suggested a portion of the overall toxicity of cyanide (CN) may be affected by nitric oxide, we investigated a long acting NO releasing complex (diethylamine/nitric oxide (DEA/NO)) which may exhibit vasodilatory as well as other nitric oxide effects to determine its ability to modify CN toxicity. Sodium nitrite, a vasodilator commonly used to treat cyanide toxicity thought to act by methemoglobin (MHb) formation, can be rapidly transformed to nitric oxide (NO). 2. Mice (n = 10 per dose) were administered one of five doses of sodium cyanide (NaCN) intraperitoneally (4.28, 5.08, 6.03, 7.17 and 8.52 mg kg-1). DEA/NO was given intravenously (20 mg kg-1) 2 min prior to NaCN. As a control, NG-monomethyl-L-arginine (L-NMMA), which inhibits NO synthesis, was administered intravenously (70 mg kg-1) to mice, 3 min prior to DEA/NO. 3. Before CN toxicity studies, we determined whether DEA/NO was producing MHb by collecting tail vein blood from mice and measuring MHb levels. For example, 4 min after DEA/NO administration (5, 10, and 20 mg kg-1), MHb levels were 1.27 +/- 0.28%, 2.60 +/- 0.26% and 6.53 +/- 0.54% respectively. O2 capacity was also decreased in a dose related manner. Carboxyhemoglobin or percent O2 saturation, on the other hand, was not significantly inhibited. The LD50 increased from 5.75 +/- 0.026 (CN alone) to 7.66 +/- 0.021 mg kg-1 (CN+DEA/NO) resulting in a protective ratio of 1.73. 4. Results suggest the following: (1) L-NMMA, which inhibits the synthesis of endogenous NO, appears to exacerbate the DEA/NO (or exogenous NO) response; (2) DEA/NO appears to reduce the toxicity of CN which suggests that a portion of CN toxicity may be affected by a NO component; and (3) low DEA/NO doses may act via a direct effect while higher doses (40 mg kg-1) may allow for formation of a concentration of MHb which can bind CN to form cyanomethemoglobin and reduce the toxicity of CN.

The effect of three alpha-keto acids on 3-mercaptopyruvate sulfurtransferase activity.

3-Mercaptopyruvate sulfurtransferase catalyzes the transfer of sulfur from 3-mercaptopyruvate to several possible acceptor molecules, one of which is cyanide. Because the transsulfuration of cyanide is the primary in vivo mechanism of detoxification, 3-mercaptopyruvate sulfurtransferase may function in the enzymatic detoxification of cyanide in vivo. Three alpha-keto acids (alpha-ketobutyrate, alpha-ketoglutarate, and pyruvate) have previously been demonstrated to be cyanide antidotes in vivo, and it has been suggested that this is due to the nonenzymatic binding of cyanide by the alpha-keto acid. However, it has also been proposed that alpha-keto acids may increase the activity of enzymes involved in the transsulfuration of cyanide. Thus, the effect of these three alpha-keto acids on the enzyme 3-mercaptopyruvate sulfurtransferase was examined. All three alpha-keto acids inhibited 3-mercaptopyruvate sulfurtransferase in a concentration-dependent manner and were determined to be uncompetitive inhibitors of MST with respect to 3-mercaptopyruvate. The inhibitor constant Ki was estimated by two methods for each inhibitor and ranged from 4.3 to 6.3 mM. The I50, which is the inhibitor concentration that produces 50% inhibition, was calculated for all three alpha-keto acids and ranged between 9.5 and 13.7 mM. These observations add further support to the hypothesis that the mechanism of the alpha-keto acid antidotes is the nonenzymatic binding of cyanide, not stimulation of enzymes involved in the transsulfuration of cyanide to thiocyanate.

Specificity studies of 3-Mercaptopyruvate sulfurtransferase.

3-Mercaptopyruvate sulfurtransferase (E.C. 2.8.1.2; MST) is an enzyme believed to function in the endogenous cyanide (CN) detoxification system because it is capable of transferring sulfur from 3-mercaptopyruvate (3-MP) to CN, forming the less toxic thiocyanate (SCN). To date, 3-MP is the only known sulfur-donor substrate for MST. In an effort to increase the understanding of what chemical properties of 3-MP affect its utilization as a substrate, in vitro enzyme kinetic studies of MST were conducted using two mercaptic acids that are structurally related to 3-MP. Neither of these compounds was able to serve as a sulfur-donor substrate for MST. Inhibitor studies determined that 3-mercaptopropionic acid did not affect the Km of MST for 3-MP but did decrease Vmax and, thus, was determined to be a noncompetitive inhibitor. Alternatively, 2-mercaptopropionic acid 2-MPA decreased Km and Vmax and was determined to be an uncompetitive inhibitor of MST with respect to 3-MP. These data indicate that the alpha-keto group of 3-MP is necessary for its utilization as a substrate, and the inhibitor studies suggest that the position of the sulfur may also affect the binding of these compounds to the enzyme. These observations increase the understanding of what factors can affect the utilization of a compound as a sulfur-donor substrate for MST and may aid in the development of alternative sulfur-donor substrates for MST.

A protein kinase C inhibitor attenuates cyanide toxicity in vivo.

We have examined the effect of pretreatment with a potent protein kinase C (PKC) inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), against metabolic alterations induced by sodium cyanide (NaCN), 4.2 mg/kg, in brain of anesthetized male micropigs (6-10 kg). Brain high energy phosphates were analyzed using a 31P nuclear magnetic resonance (NMR) spectroscopic surface coil in a 4.7 Telsa horizontal bore magnet. H-7, 1 mg/kg, was given intravenously (i.v.) 30 min before NaCN challenge (H-7 + CN-). Prior to NaCN, H-7, or H-7 + CN- administration, baseline 31P resonance spectra of 1-min duration were acquired for 5-10 min, and continued for an additional 60 min following i.v. NaCN injection, each animal serving as its own control. Peaks were identified as phosphomonoester (PME), inorganic phosphate (Pi), phosphodiester (PDE), phosphocreatine (PCr) and adenosine triphosphate (ATP), based on their respective chemical shifts. Without H-7 pretreatment, NaCN effects were marked by a rising Pi and a declining PCr peak 2 min after injection, with only 2/5 of the animals surviving the 60 min experiment. Through a pretreatment period of 30 min, H-7 did not affect baseline cell energy profile as reflected by the 31P-NMR spectra, but in its presence, those changes (i.e. diminishing PCr and rising Pi peaks) elicited by NaCN were markedly blunted; 4/5 of the animals in this group survived the NaCN challenge. It is proposed that H-7, a pharmacologic inhibitor of PKC, may be useful in CN- antagonism, underscoring the role of PKC in cyanide intoxication.

Topical anesthetic-induced methemoglobinemia and sulfhemoglobinemia in macaques: a comparison of benzocaine and lidocaine.

Benzocaine (BNZ) and lidocaine (LC) are commonly used topical (spray) anesthetics approved for use in humans. Benzocaine has structural similarities to methemoglobin (MHb)-forming drugs that are current candidates for cyanide prophylaxis, while LC has been reported to increase MHb in man. In this study, we compared MHb and sulfhemoglobin (SHb) production in three groups of Macaques (Chinese rhesus and Indian rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina)) after exposure to BNZ and LC. Formation of SHb, unlike MHb, is not thought to be reversible and therefore is considered to be of greater toxic significance. Both MHb and SHb levels were measured periodically on a CO-Oximeter. All rhesus macaques (n = 8) were administered an intratracheal/intranasal) dose of 56 mg (low dose) or 280 mg (high dose) of BNZ or 40 mg of LC in a randomized cross-over design (all animals received all three treatments). Pig-tailed macaques (n = 6) were given an intranasal dose of 56 mg of BNZ and 40 mg of LC. As no differences in the peak MHb or time to peak (mean +/- SD) were observed among the three macaque subspecies, the data were pooled. Lidocaine did not cause MHb or SHb formation above baseline in any monkey. In contrast, all monkeys (n = 14) had a significant elevation in peak MHb formation after 56 mg of BNZ, which ranged from 4.0% to 19.4% with an average of 8.6 +/- 4.0% (mean +/- SD), with peak MHb levels reached at 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C modulation of rhodanese-catalyzed conversion of cyanide to thiocyanate.

Detoxification of cyanide is catalyzed by a sulfurtransferase, rhodanese, a phosphoprotein regulated by unknown protein kinases. In this study, we determined if a Ca2+/phospholipid-modulated phosphotransferase, protein kinase C (PKC) could modify rhodanese activity. Thiocyanate (SCN-) production as an estimate of rhodanese activity in vitro was measured in the presence or absence of exogenously added purified PKC, or 12-O-tetradecanoylphorbol acetate (TPA), a pharmacologic activator of the endogenous PKC. HI-6 (1-(2-(hydroximino)methyl))pyridinium-2-(4-(aminocarbonyl) pyridinium dimethylether) is an oxime that may dephosphorylate phosphoproteins due to the proposed phosphatase-like activity of the oximes. We examined HI-6's effect on rhodanese-catalyzed SCN- production. Bovine kidney rhodanese (0.40 mg/ml protein) was reacted with 4 mM KCN and SCN- production determined spectrophotometrically following the method of Westley (1981). Preincubating rhodanese with 20 or 100 ng of purified PKC (alpha, beta, gamma isozymes) for 5 min before initiating the reaction with 4 mM KCN as the substrate increased SCN- production by 17 or 40%, respectively, over the control (P < 0.05). Rhodanese formation of SCN- decreased when the preincubation was conducted with 1 nM or 100 nM of TPA. With HI-6 at 1 or 10 microM used in place of PKC, or TPA, rhodanese activity was increased by 6 or 14% (P < 0.05), respectively, compared to control. Under the conditions examined, exogenous PKC acting as a possible phosphate acceptor, and HI-6, a potential dephosphorylating compound, increased rhodanese activity. These data are consistent with the observation that rhodanese can exist as a phosphorylated enzyme which is not active and a dephosphorylated form which is active. It is suggested that addition of purified, exogenous PKC may accept phosphate from phosphorylated rhodanese or HI-6 may dephosphorylate rhodanese, both of which stimulate the conversion of cyanide anion to the less toxic SCN-. These observations support the possibility that rhodanese may be regulated by protein phosphorylation and treatments that alter the phosphorylation state of rhodanese may affect cyanide detoxification via SCN- formation.

The antidotal action of sodium nitrite and sodium thiosulfate against cyanide poisoning.

The combination of sodium thiosulfate and sodium nitrite has been used in the United States since the 1930s as the primary antidote for cyanide intoxication. Although this combination was shown to exhibit much greater efficacy than either ingredient alone, the two compounds could not be used prophylactically because each exhibits a number of side effects. This review discusses the pharmacodynamics, pharmacokinetics, and toxicology of the individual agents, and their combination.