Herbert T. Nagasawa

Mechanisms of Inhibition of Aldehyde Dehydrogenase by Nitroxyl, the Active Metabolite of the Alcohol Deterrent Agent Cyanamide

Nitroxyl, produced in the bioactivation of the alcohol deterrent agent cyanamide, is a potent inhibitor of aldehyde dehydrogenase (AIDH); however, the mechanism of inhibition of AlDH by nitroxyl has not been described previously. Nitroxyl is also generated from Angelis salt (Na2N2O3) at physiological pH, and, indeed, Angelis salt inhibited yeast AlDH in a time- and concentration-dependent manner, with IC50 values under anaerobic conditions with and without NAD+ of 1.3 and 1.8 microM, respectively. Benzaldehyde, a substrate for AlDH, competitively blocked the inhibition of this enzyme by nitroxyl in the presence of NAD+, but not in its absence, in accord with the ordered mechanism of this reaction. The sulfhydryl reagents dithiothreitol (5 mM) and reduced glutathione (10 mM) completely blocked the inhibition of AlDH by Angelis salt. These thiols were also able to partially restore activity to the nitroxyl-inhibited enzyme, the extent of reactivation being dependent on the pH at which the inactivation occurred. This pH dependency indicates the formation of two inhibited forms of the enzyme, with an irreversible form predominant at pH 7.5 and below, and a reversible form predominant at pH 8.5 and above. The reversible form of the inhibited enzyme is postulated to be an intra-subunit disulfide, while the irreversible form is postulated to be a sulfinamide. Both forms of the inhibited enzyme are derived via a common N-hydroxysulfenamide intermediate produced by the addition of nitroxyl to active site cysteine thiol(s).

Donors of HNO.

Recent comparisons of the pharmacological effects of nitric oxide (NO) and nitroxyl (HNO) donors have demonstrated that the responses to these redox-related nitrogen oxides are nearly universally dissimilar. These analyses have suggested the existence of mutually exclusive signaling pathways as a result of discrete chemical interactions of HNO and NO with a variety of critical biomolecules. Although the mechanisms of action are currently unresolved, the pharmacological responses to HNO are promising for clinical treatment of cardiovascular diseases such as heart failure, myocardial infarction and stroke. This review provides a detailed discussion of the most commonly utilized donors of HNO as well as a guideline for the characterization of novel donors.

The metabolic activation of cyanamide to an inhibitor of aldehyde dehydrogenase is catalyzed by catalase

The inhibition of aldehyde dehydrogenase by cyanamide is dependent on an enzyme catalyzed conversion of the latter to an active metabolite. The following results suggest that catalase is the enzyme responsible for this bioactivation. The elevation of blood acetaldehyde elicited by cyanamide after ethanol administration to rats was attenuated more than 90 percent by pretreatment with the catalase inhibitor, 3-amino-1,2,4-triazole. This attenuation was dose dependent and was accompanied by a reduction in total hepatic catalase activity. Although hepatic catalase was also inhibited by cyanamide, a positive correlation between blood acetaldehyde and hepatic catalase activity was observed. In vitro, the activation inhibitor, 3-amino-1,2,4-triazole. This attenuation was dose dependent and was accompanied by a reduction in total hepatic catalase activity. Although hepatic catalase was also inhibited by cyanamide, a positive correlation between blood acetaldehyde and hepatic catalase activity was observed. In vitro, the activation of cyanamide was catalyzed by a) the rat liver mitochondrial subcellular fraction, b) the 50-65% ammonium sulfate mitochondrial fraction and c) purified bovine liver catalase. Cyanamide activation was inhibited by sodium azide. Since much of the hepatic catalase is localized in the peroxisomes and since peroxisomes and mitochondria cosediment, the cyanamide activating enzyme, catalase, is likely of peroxisomal and mitochondrial origin.

Lowering of ethanol-derived circulating blood acetaldehyde in rats by D-penicillamine

Abstract The sulfhydryl amino acid, D-penicillamine, but not L-cysteine or L-cystine, when administered to disulfiram-treated rats 1 hour before a dose of ethanol lowered the ethanol-derived, circulating blood acetaldehyde to 10% of control values. This was accompanied by a concomitant lowering of AcH in the expired air of penicillamine-treated rats. Since blood ethanol levels were the same in saline injected controls and in sulfhydryl amino acid-treated rats, this lowering of blood acetaldehyde was not due to any malabsorption of ethanol or to inhibition of the enzyme(s) that metabolize ethanol. By administration of D-penicillamine in multiple, divided doses, blood acetaldehyde generated during ethanol metabolism was reduced an average of 70% over an 8 hour period.

Differential inhibition of rat tissue catalase by cyanamide

The relative sensitivity of rat tissue catalase to inhibition by intraperitoneally administered cyanamide was liver greater than kidney greater than heart greater than brain, whereas the activity of the erythrocyte enzyme was affected minimally. The measured ED50 values for cyanamide in these tissues were 31, 44, 107 and 680 mumoles/kg body weight for liver, kidney, heart and brain respectively. On a molar basis, cyanamide was approximately twenty times more potent than 3-amino-1,2,4-triazole (3-AT) in inhibiting hepatic catalase in vivo in the rat. Like 3-AT, cyanamide inhibited erythrocyte catalase activity in vitro in the presence of hydrogen peroxide. The apparent similarities between the inhibition of hepatic catalase by cyanamide and 3-AT in vivo suggest that cyanamide belongs to the family of 3-AT-like catalase inhibitors.

Glutathione monoethyl ester protects against glutathione deficiencies due to aging and acetaminophen in mice

Our previous results indicated that glutathione (GSH) and/or cysteine (Cys) deficiency occurs in many aging tissues and also after acetaminophen (APAP) administration. The aim of this study was to investigate whether GSH monoethyl ester (GSH-OEt) can correct these deficiencies. Mice of different ages (3-31 months) through the life span were sacrificed 2 h after i.p. injection of GSH-OEt (10 mmol/kg). In separate experiments, old mice (30-31 months) received the same dose of ester 30 min before the administration of APAP (375 mg/kg) or buthionine sulfoximine (BSO, 4 mmol/kg), an inhibitor of GSH synthesis. Liver and kidney samples were analyzed for GSH and Cys by HPLC. The hepatic GSH and renal cortical GSH and Cys concentrations were about 30% lower in old mice (30-31 months) compared to mature mice (12 months). GSH-OEt corrected these aging-related decreases. APAP decreased both hepatic and renal cortical GSH and Cys concentrations in old mice, but GSH-OEt prevented these decreases. GSH-OEt also prevented the BSO-induced decreases in hepatic and renal GSH concentrations. The results demonstrated that GSH-OEt protected against GSH deficiency due to biological aging as well as APAP-induced decreases in old mice.

Mechanism for the Inhibition of Aldehyde Dehydrogenase by Nitric Oxide

The inhibition of Saccharomyces cerevisiae aldehyde dehydrogenase (AlDH) by gaseous nitric oxide (NO) in solution and by NO generated from diethylamine nonoate was time and concentration dependent. The presence of oxygen significantly reduced the extent of inhibition by NO, indicating that NO itself rather than an oxidation product of NO such as N2O3 is the inhibitory species under physiological conditions. A cysteine residue at the active site of the enzyme was implicated in this inhibition based on the following observations: a) NAD+ and NADP+, but not reduced cofactors, significantly enhanced inhibition of AlDH by NO; b) the aldehyde substrate, benzaldehyde, blocked inhibition; and c) inhibition was accompanied by loss of free sulfhydryl groups on the enzyme. Activity of the NO-inactivated enzyme was readily restored by treatment with dithiothreitol (DTT), but not with GSH. This difference was attributed, in part, to a redox process leading to the formation of a cyclic DTT disulfide. Based on the chemistry deduced from model systems, the reaction of NO with AlDH sulfhydryls was shown to produce intramolecular disulfides and N2O. These disulfides were shown to be intrasubunit disulfides by nonreducing SDS-PAGE analysis of the NO- inhibited enzyme. Following complete inhibition of AlDH by NO, four of the eight titratable (Ellmans reagent) sulfhydryl groups of AlDH were found to be oxidized to disulfides. These results suggest that a) the sulfhydryl group of active site Cys-302 and a proximal cysteine are oxidized to form an intrasubunit disulfide by NO; b) only two of the four subunits of AlDH are catalytically active; and c) NO preferentially oxidizes sulfhydryl groups of the catalytically active subunits. A detailed mechanism for the inhibition of AlDH by NO is presented.

Sulfanegen sodium treatment in a rabbit model of sub-lethal cyanide toxicity

The aim of this study is to investigate the ability of intramuscular and intravenous sulfanegen sodium treatment to reverse cyanide effects in a rabbit model as a potential treatment for mass casualty resulting from cyanide exposure. Cyanide poisoning is a serious chemical threat from accidental or intentional exposures. Current cyanide exposure treatments, including direct binding agents, methemoglobin donors, and sulfur donors, have several limitations. Non-rhodanese mediated sulfur transferase pathways, including 3-mercaptopyruvate sulfurtransferase (3-MPST) catalyze the transfer of sulfur from 3-MP to cyanide, forming pyruvate and less toxic thiocyanate. We developed a water-soluble 3-MP prodrug, 3-mercaptopyruvatedithiane (sulfanegen sodium), with the potential to provide a continuous supply of substrate for CN detoxification. In addition to developing a mass casualty cyanide reversal agent, methods are needed to rapidly and reliably diagnose and monitor cyanide poisoning and reversal. We use non-invasive technology, diffuse optical spectroscopy (DOS) and continuous wave near infrared spectroscopy (CWNIRS) to monitor physiologic changes associated with cyanide exposure and reversal. A total of 35 animals were studied. Sulfanegen sodium was shown to reverse the effects of cyanide exposure on oxyhemoglobin and deoxyhemoglobin rapidly, significantly faster than control animals when administered by intravenous or intramuscular routes. RBC cyanide levels also returned to normal faster following both intramuscular and intravenous sulfanegen sodium treatment than controls. These studies demonstrate the clinical potential for the novel approach of supplying substrate for non-rhodanese mediated sulfur transferase pathways for cyanide detoxification. DOS and CWNIRS demonstrated their usefulness in optimizing the dose of sulfanegen sodium treatment.

N,O-Diacylated-N-hydroxyarylsulfonamides: Nitroxyl precursors with potent smooth muscle relaxant properties

N,O-Diacylated-N-hydroxyarylsulfonamides are capable of slowly releasing nitroxyl (HNO) by simple, non-enzymatic hydrolysis in Krebs solution at 37 degrees C. Release of nitric oxide (NO) was not seen. These compounds were also found to elicit vasorelaxation in rabbit thoracic aorta in vitro, presumably as a result of their ability to release HNO. This effect was enhanced by the addition of superoxide dismutase (SOD). Thus, these results are consistent with previous work indicating that HNO is a potent vasorelaxant.

Metabolic activation of cyanamide by liver mitochondria, a requirement for the inhibition of aldehyde dehydrogenase enzymes.

The possibility that the cyanamide inhibition of aldehyde dehydrogenase in vivo may be dependent on the conversion of cyanamide to an active form was assessed. Yeast aldehyde dehydrogenase and cyanamide were incubated in the presence and absence of intact rat liver mitochondria. The yeast enzyme was completely inhibited in systems containing both cyanamide (200 μM) and mitochondria, whereas no significant loss of enzyme activity was observed with up to 10 mM cyanamide without added mitochondria. Similar results were obtained using rabbit skeletal muscle glyceraldehyde-3-phosphate dehydrogenase. In the presence of mitochondria, the I 50 for the inhibition of the yeast enzyme by cyanamide was 7.8 μM. This inhibition was time-dependent and dependent on the concentration of mitochondrial protein. Heat denatured mitochondria did not activate cyanamide. These results demonstrate that the inhibition of aldehyde dehydrogenase by cyanamide is dependent on its conversion to an active form, and intact rat liver mitochondria can catalyze this metabolic activation.