Harriet Kruszyna

The effect of methemoglogin on the inhibition of cytochrome c oxidase by cyanide, sulfide or azide

Abstract Under our experimental conditions sulfide was a more potent inhibitor of a particulate preparation of cytochrome oxidase than was cyanide; azide proved to be a relatively weak inhibitor, all of which is in agreement with the observations of others. The undissociated species (H2S) appeared to be more inhibitory than the anionic species (HS−) in accord with the conclusions of others about HCN and HN3. Addition of methemoglobin to the oxidase inhibited by cyanide or sulfide restored the activity of the enzyme system, but the addition of methemoglobin to the azide-inhibited oxidase under the same conditions had little or no effect. Our results suggest that sulfide produces death in animals by inhibition of cytochrome oxidase, but such a mechanism seems unlikely in the case of azide.

Nitric oxide hemoglobin in mice and rats in endotoxic shock

Mice given ip bacterial endotoxin (LPS) at 10 mg/kg showed a statistically significant decrease in plasma glucose and an increase in hematocrit at 2 h after injection. Glucose was still decreased at 4 h, but the hematocrit had returned to control values. Nitrosylated hemoglobin (HbNO) was detected at 3, but not at 2 h. By 4 h it had increased 5-fold. When N-monomethylarginine (NMMA) at 100 mg/kg, ip was given 2 h after LPS in mice, the HbNO concentration at 4 h was significantly reduced, but the hypoglycemia was worsened because NMMA itself produced a significant hypoglycemia. Rats given iv LPS, 20 mg/kg, showed a fleeting, transient rise in mean arterial pressure (MAP) lasting only a few min. Thereafter, the MAP tended to drift slowly downward over 4 h, but when the MAP at 30 min intervals was compared to the pre-LPS MAP, there were no significant differences. Plasma glucose in unanesthetized rats was significantly elevated at 1 h, back to control at 2 h, and significantly decreased at 3 h. HbNO was detected as early as 1 h after injection. By 2 h the HbNO concentrations exceeded the highest levels found in mice, and they were still increasing as late as 5 h after injection. Unanesthetized rats showed toxic signs and 3/12 rats died within 4 hours of LPS administration. These results are consistent with a model for endotoxic shock in which LPS stimulates an inducible pathway for NO synthesis.

Red blood cells generate nitric oxide from directly acting, nitrogenous vasodilators

Human red blood cells (RBC) incubated under nitrogen with methylene blue and glucose at physiological temperature and pH can be used to test for the biotransformation of nitrogenous vasodilators to nitric oxide (NO). The NO generated was trapped as nitrosylated heme by reduced subunits (hemeII) on various hemoglobin valency species and quantified by electron paramagnetic resonance spectroscopy. It was possible to separate the various valency species of hemoglobin present in the mixture as (alpha 2 + beta 2)2, (alpha 2 + beta 3+)2, (alpha 3 + beta 2+)2, or (alpha 3 + beta 3+)2 by isoelectric focusing (IEF) unless cyanide (from nitroprusside) or azide was present in the mixture. These anions bind tenaciously to oxidized subunits (hemeIII) and prevent the separation of the various species by IEF. The fully oxidized tetramer, (alpha 3 + beta 3+)2, does not bind NO, but the other three species have hemeII units which can be nitrosylated. In the absence of cyanide or azide the valency species could be separated by IEF, and it was possible to quantify the degree of nitrosylation on each individual species. The various agents tested (nitrite, glyceryl trinitrate, hydroxylamine, hydralazine, nitroprusside, and azide) produced different patterns of valency species and degrees of nitrosylation of hemeII. When hemeIII ligands were present or in cases of very low yields, it was still possible to quantify the total concentration of NO-hemeII in the mixture. Thus, the method could still be used to test for NO formation. All of the so-called NO vasodilators tested yielded detectable amounts of NO in the system.

Acute Neurotoxicity of Sodium Azide and Nitric Oxide

Sodium azide is a chemical of rapidly growing commercial importance with a high acute toxicity and an unknown mechanism of action. Although it has some chemical properties and biological effects in common with cyanide, its lethality does not appear to be due to inhibition of cytochrome oxidase. Unlike cyanide it is a potent vasodilator and inhibitor of platelet aggregation presumably by virtue of its conversion to nitric oxide in vivo and in isolated preparations of blood vessels and thrombocytes. It is not clear whether the high toxicity of azide is due to nitric oxide or to the parent anion. Of a number of possible azide antagonists tested in intact mice only phenobarbital in both anesthetic and subanesthetic doses afforded statistically significant protection against death. Diazepam, phenytoin, and an anesthetic dose of a ketamine/xylazine combination had no effect. Major motor seizures are sometimes seen in human azide poisoning, and these are a regular feature of azide poisoning in laboratory rodents. Solutions of nitric oxide given systemically to mice produced no signs of toxicity, but doses 1,000-fold lower placed in the cerebroventricular system of rats produced brief but violent tonic convulsive episodes. A dose of 0.61 mmol/kg azide as given systemically regularly produced convulsions whereas a dose of 6 mumol/kg given icv produced seizures in rats. The icv convulsive dose of azide was 50-fold larger than the icv dose of nitric oxide. These results suggest that azide lethality is due to enhanced excitatory transmission in the central nervous system perhaps after its conversion to nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of valency hybrids and nitrosylated species of hemoglobin in mice by nitric oxide vasodilators.

The hemoglobin fraction of blood samples from C57BL/6 male mice was purified by column chromatography and subjected to isolectric focusing (IEF) across a pH gradient. Densitometric scanning of the IEF gel showed the presence of a single peak corresponding to the fully reduced tetramer, H, or (alpha 2+ beta 2+)2. Twenty minutes after an ip injection of 1.1 mmol/kg NaNO2 blood samples treated the same way showed four peaks corresponding to the species: H = 43%, X or (alpha 2+ beta 3+)2 = 10%, Y or (alpha 3+ beta 2+)2 = 33%, and M or (alpha 3+ beta 3+)2 = 14%. In contrast blood samples from control CD-1 male mice showed the presence of three IEF distinct peaks which were all believed to be H valency forms, and six distinct peaks were seen after treatment in vivo with NaNO2. Thus, the C57BL/6 mice yield patterns similar to those observed after in vitro treatment of human red cells with NaNO2 (H. Kruszyna, R. Kruszyna, R. P. Smith, and D. E. Wilcox, 1987b, Toxicol. Appl. Pharmacol. 91, 429-438), and the CD-1 mice are a much less satisfactory model. The appearance and disappearance of the species X, Y, and M over time after ip injection of 1.1 mmol/kg NaNO2 or hydroxylamine HCl were followed in C57BL/6 mice by the technique of IEF. In each case the patterns were consistent with previously established patterns for the respective methemoglobinemias as determined by absorption spectrophotometry, and they were consistent with the suggestion that two pathways exist for the oxidation of H and for the reduction of M which proceed through X and Y, respectively. By using electron paramagnetic resonance (EPR) spectroscopy, we were also able to follow with time the concentration of nitrosylated heme (NO-heme) on reduced subunits in both mouse strains. The peak for the NO-heme coincided in time with the peak methemoglobinemia as determined by either IEF or absorption spectrophotometry. EPR was also used to determine NO-heme in CD-1 mice after injection of a series of NO-vasodilators with and without methylene blue (MB). Low, but clearly detectable amounts of NO-heme were found in the blood of animals given all xenobiotics tested including NaNO2, hydroxylamine HCl, glyceryl trinitrate, hydralazine, sodium nitroprusside, and sodium azide. MB has little effect on the response, and no NO-heme could be detected in control mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of hydroxylamine, 4-dimethylaminophenol and nitrite protection against cyanide poisoning in mice

Intraperitoneal doses of 4-dimethylaminophenol hydrochloride (DMAP), hydroxylamine hydrochloride (H2NOH) and sodium nitrite (NaNO2) were found where each converted a maximum of about 37% of the total circulating hemoglobin in mice to methemoglobin. Those doses in mmol/kg were: 0.29 for DMAP, 1.1 for H2NOH, and 1.1 for NaNO2. For DMAP and H2NOH the peak was sharp and at about 7 min after injection whereas for NaNO2 the peak was much broader and at about 40 min. The i.p. LD50s in mmol/kg were: 0.48 for DMAP, 1.8 for H2NOH and 2.3 for NaNO2. When mice pretreated with each of the methemoglobin-generating agents were challenged with sodium cyanide, the ratios of the LD50s in protected mice to those in control mice (protection index, PI) were 1.5 for H2NOH, 2.0 for DMAP and 3.1 for NaNO2. When sodium thiosulfate was also given in combination with each of the three methemoglobin-generating agents, the protective effect was at least additive. The PI against sodium sulfide was also significantly greater in mice pretreated with NaNO2 than in mice given H2NOH. Methemoglobins generated from human and mouse hemoglobins by either NaNO2 or by H2NOH had identical binding affinities (dissociation constants) for cyanide. When human red cells containing methemoglobin generated by exposure to either NaNO2 or H2NOH were injected into the peritoneal cavity of mice and then followed by cyanide challenges, there was no difference in the PI for the two kinds of methemoglobin. Not only was the PI the same in each case with human cells, but it was also identical with that in mice given NaNO2 systemically to generate the same total amount of methemoglobin. The difference in PI between NaNO2 and H2NOH (or DMAP) in mice appears to be related to the high rate of methemoglobin reductase activity in mouse RBC. It appears likely that cyanmethemoglobin is a substrate for mouse methemoglobin reductase activity, and that NaNO2 is an inhibitor of mouse methemoglobin reductase. No differences in cyanide antagonism between NaNO2 and H2NOH would be anticipated in humans because of the slow rates of methemoglobin reduction in human red cells.

Cyanide and sulfide interact with nitrogenous compounds to influence the relaxation of various smooth muscles

Abstract Sodium nitroprusside relaxed guinea pig ileum after the segment had been submaximally contracted by either histamine or acetylcholine, intact isolated rabbit gall bladder after submaximal contraction by either acetylcholine or cholecystokinin octapeptide, and rat pulmonary artery helical strips after submaximal contraction with norepinephrine. In each of these cases the relaxation produced by nitroprusside was at least partially reversed by the subsequent addition of excess sodium cyanide. Cyanide, however, in nontoxic concentrations did not reverse the spasmolytic effects of hydroxylamine hydrochloride, sodium azide, nitroglycerin, sodium nitrite, or nitric oxide hemoglobin on guinea pig ileum, nor did cyanide alone in the same concentrations have any effect. The similar interaction between nitroprusside and cyanide on rabbit aortic strips is not dependent on the presence of an intact endothelial cell layer. Also, on rabbit aortic strips and like cyanide, sodium sulfide reversed the spasmolytic effects of azide and hydroxylamine, but it had little or no effect on the relaxation induced by papaverine. Unlike cyanide, however, sulfide augmented the relaxation induced by introprusside, and it reversed the effects of nitric oxide hemoglobin, nitroglycerin, and nitrite. A direct chemical reaction between sulfide and nitroprusside may account for the difference between it and cyanide. Although evidence was obtained also for a direct chemical reaction between sulfide and norepinephrine, that reaction does not seem to have played a role in these results. These observations suggest the existence of at least three distinct subclasses of so-called nitric oxide vasodilators. At least in some cases cyanide and sulfide cannot be acting by the same mechanism in their modifications of the responses to the agonists.

Nitroprusside Increases Cyclic Guanylate Monophosphate Concentrations during Relaxation of Rabbit Aortic Strips and Both Effects Are Antagonized by Cyanide

The authors have confirmed previous observations that sodium cyanide (CN-) partially reverses the vasodilator effects of sodium nitroprusside (SNP) on vascular smooth muscle. As tested on rabbit aortic strips contracted by norepinephrine (NE), the final tension is independent of the order of addition of reagents. In the same concentration, CN- alone had no effect on tension also as reported by others. The ED50 values for relaxation of aortic strips for a series of directly acting agonists (“nitric oxide vasodilators”) were: sodium azide (N-3) 2.1 X 10-7 M; SNP 2.7 X 10-7 M; hydroxylamine (H2NOH) hydrochloride 2.5 X 10-6 M; human nitric oxide hemoglobin (HbNO) 3.5 X 10-6 M; and sodium nitrite (NO-2) 1.2 X 10-4 M. In addition to SNP, CN- antagonized the vasodilator effects of N-3 and H2NOH, but it failed to reverse relaxation by HbNO, NO gas, NO-2 (as observed by us), glyceryl trinitrate, adenosine, or papaverine (as observed by others). The only change noted in cyclic-adenosine monophosphate (c-AMP) concentrations in aortic strips exposed to 1) NE, 2) NE + NO-2 or SNP, or 3) NE + NO-2 or SNP + CN- was an increase due to NE. The only statistically significant change noted in cyclicguanosine monophosphate (c-GMP) concentrations exposed to 1) NE, 2) NE + NO-2) or 3) NE + NO-2 + CN- was also an increase due to NE. In contrast, SNP resulted in further increases in c-GMP after NE, and when cyanide was added, a significant decrease in c-GMP followed. These results are only partially consistent with a role for c-GMP in relaxation of vascular smooth muscle, but cyanide may become a useful tool for the study of mechanisms of action of the nitric oxide vasodilators.

DETERMINING SODIUM AZIDE CONCENTRATION IN BLOOD BY ION CHROMATOGRAPHY

We describe a simple method for measuring sodium azide concentrations in aliquots of blood and other tissues. Aliquots are acidified, converting azide to volatile hydrazoic acid (HN3) which is then trapped in sodium hydroxide. We analyze the resulting aliquots by ion chromatography, using a sodium tetraborate eluent and suppressed conductivity detection. The method is sensitive to at least 100 ng/mL.

Nitroprusside: a potpourri of biologically reactive intermediates.

Sodium nitroprusside, Na2[Fe(CN)5NO], (SNP) has been recognized as a potent, directly acting vasodilator for over a half-century. More recently, it has been found also to inhibit blood platelet aggregation and adhesion. These biologicial activities are ascribed to the nitrosyl ligand on SNP which is thought to activate guanylate cyclase by binding to its critical heme group. c-GMP in turn initiates a cascade of kinase reactions which result in the utimate biological effects. Thus, SNP is one of the compounds known as the nitric oxide (NO) vasodilators (Ignarro, 1989) which mimic the effects of the so-called endothelium-derived relaxing factor (Furchgott and Zawadzki, 1980), an endogenous mediator which many are convinced is identical to NO. The same or a similar factor is believed to play in role in excitatory amino acid transmission in the central nervous system, and to be the cytotoxic factor synthesized by neutrophils and macrophages (Collier and Valiance, 1989).