Prevention of naphthalene-induced pulmonary toxicity by glutathione prodrugs: roles for glutathione depletion in adduct formation and cell injury.

Naphthalene is metabolized in the lung and liver to reactive intermediates by cytochrome P450 enzymes. These reactive species deplete glutathione, covalently bind to proteins, and cause necrosis in Clara cells of the lung. The importance of glutathione loss in naphthalene toxicity was investigated by using the glutathione prodrugs (glutathione monoethylester or cysteine-glutathione mixed disulfide) to maintain glutathione pools during naphthalene exposure. Mice given a single intraperitoneal injection of naphthalene (1.5 mmol/kg) were treated with either prodrug (2.5 mmol/kg) 30 min later. Both compounds effectively maintained glutathione levels and decreased naphthalene-protein adducts in the lung and liver. However, cysteine-glutathione mixed disulfide was more effective at preventing Clara cell injury. To study the prodrugs in Clara cells without the influence of hepatic naphthalene metabolism and circulating glutathione, dose-response and time-course studies were conducted with intrapulmonary airway explant cultures. Only the ester of glutathione raised GSH in vitro; however, both compounds limited protein adducts and cell necrosis. In vitro protection was not associated with decreased naphthalene metabolism. We conclude that (1) glutathione prodrugs can prevent naphthalene toxicity in Clara cells, (2) the prodrugs effectively prevent glutathione loss in vivo, and (3) cysteine-glutathione mixed disulfide prevents naphthalene injury in vitro without raising glutathione levels.

Inhibition of ALDH3A1-catalyzed oxidation by chlorpropamide analogues.

In our efforts to identify agents that would specifically inhibit ALDH3A1, we had previously studied extensively the effect of an N(1)-alkyl, an N(1)-methoxy, and several N(1)-hydroxy-substituted ester derivatives of chlorpropamide on the catalytic activities of ALDH3A1s derived from human normal stomach mucosa (nALDH3A1) and human tumor cells (tALDH3A1), and of two recombinant aldehyde dehydrogenases, viz. human rALDH1A1 and rALDH2. The N(1)-methoxy analogue of chlorpropamide, viz. 4-chloro-N-methoxy-N-[(propylamino)carbonyl]benzenesulfonamide (API-2), was found to be a relatively selective and potent inhibitor of tALDH3A1-catalyzed oxidation as compared to its ability to inhibit nALDH3A-catalyzed oxidation, but even more potently inhibited ALDH2-catalyzed oxidation, whereas an ester analogue, viz. (acetyloxy)[(4-chlorophenyl)sulfonyl]carbamic acid 1,1-dimethylethyl ester (NPI-2), selectively inhibited tALDH3A1-catalyzed oxidation as compared to its ability to inhibit nALDH3A1-, ALDH1A1- and ALDH2-catalyzed oxidations, and this inhibition was apparently irreversible. Three additional chlorpropamide analogues, viz. 4-chloro-N,O-bis(ethoxycarbonyl)-N-hydroxybenzenesulfonamide (NPI-4), N,O-bis(carbomethoxy)methanesulfohydroxamic acid (NPI-5), and 2-[(ethoxycarbonyl)oxy]-1,2-benzisothiazol-3(2H)-one 1,1-dioxide (NPI-6), were evaluated in the present investigation. Quantified were NAD-linked oxidation of benzaldehyde catalyzed by nALDH3A1 and tALDH3A1, and NAD-linked oxidation of acetaldehyde catalyzed by rALDH1A1 and rALDH2, all at 37 degrees C and pH 8.1, and in the presence and absence of inhibitor. NPI-4, NPI-5 and NPI-6 were not substrates for the oxidative reactions catalyzed by any of the ALDHs studied. Oxidative reactions catalyzed by the ALDH3A1s, rALDH1A1 and rALDH2 were each inhibited by NPI-4 and NPI-5. NPI-6 was a poor inhibitor of nALDH3A1- and tALDH3A1-catalyzed oxidations, but was a relatively potent inhibitor of rALDH1A1- and rALDH2-catalyzed oxidations. In all cases, inhibition of ALDH-catalyzed oxidation was directly related to the product of inhibitor concentration and preincubation (enzyme+inhibitor) time. As judged by the product values (microM x min) required to effect 50% inhibition (IC(50)): (1) nALDH3A1 and tALDH3A1 were essentially equisensitive to inhibition by NPI-4 and NPI-5, and both enzymes were poorly inhibited by NPI-6; (2) rALDH1A1 was, relative to the ALDH3A1s, slightly more sensitive to inhibition by NPI-4 and NPI-5, and far more sensitive to inhibition by NPI-6; and (3) rALDH1A1 was, relative to rALDH2, essentially equisensitive to inhibition by NPI-5, whereas, it was slightly more sensitive to inhibition by NPI-4 and NPI-6.

N-hydroxybenzenecarboximidic acid derivatives: a new class of nitroxyl-generating prodrugs.

On the basis of the propensity of Piloty's acid to generate nitroxyl (HNO), we previously prepared a number of N,O-bisacylated Piloty's acid derivatives and showed that such prodrugs underwent a disproportionation reaction following ester hydrolysis to give an unstable intermediate that hydrolyzed to nitroxyl. To expand the versatility of this series, we desired some mixed N,O-diacylated Piloty's acid derivatives and devised a synthetic route to them. Such efforts led us, serendipitously, to a new series of heretofore unreported nitroxyl-generating compounds. Thus, benzohydroxamic acid was acylated on the hydroxylamino oxygen and the resulting product converted to its sodium salt. Treatment of this salt with arenesulfonyl chorides would be expected to give the mixed N,O-diacylated derivatives of Piloty's acid. However, the products obtained were the isomeric carboximidic acid derivatives whose structures were deduced from the IR and (13)C NMR spectral frequencies associated with the sp(2) carbons. The structures were verified by analysis of the X-ray crystal structure of a prototype compound of this series. When incubated with porcine liver esterase or mouse plasma, these N-acyloxy-O-arenesulfonylated benzenecarboximidic acid derivatives liberated HNO, measured as N(2)O, as well as the expected arenesulfinic acid and benzoic acid. Alkaline hydrolysis also produced N(2)O, but the major products were the arenesulfonic acid and benzohydroxamic acid. Thus, these N-hydroxybenzenecarboximidic acid derivatives represent a new series of nitroxyl prodrugs that require enzymatic bioactivation before nitroxyl can be liberated.

Inhibition of ALDH3A1-catalyzed oxidation by chlorpropamide analogues.

In our efforts to identify agents that would specifically inhibit ALDH3A1, we had previously studied extensively the effect of an N(1)-alkyl, an N(1)-methoxy, and several N(1)-hydroxy-substituted ester derivatives of chlorpropamide on the catalytic activities of ALDH3A1s derived from human normal stomach mucosa (nALDH3A1) and human tumor cells (tALDH3A1), and of two recombinant aldehyde dehydrogenases, viz. human rALDH1A1 and rALDH2. The N(1)-methoxy analogue of chlorpropamide, viz. 4-chloro-N-methoxy-N-[(propylamino)carbonyl]benzenesulfonamide (API-2), was found to be a relatively selective and potent inhibitor of tALDH3A1-catalyzed oxidation as compared to its ability to inhibit nALDH3A-catalyzed oxidation, but even more potently inhibited ALDH2-catalyzed oxidation, whereas an ester analogue, viz. (acetyloxy)[(4-chlorophenyl)sulfonyl]carbamic acid 1,1-dimethylethyl ester (NPI-2), selectively inhibited tALDH3A1-catalyzed oxidation as compared to its ability to inhibit nALDH3A1-, ALDH1A1- and ALDH2-catalyzed oxidations, and this inhibition was apparently irreversible. Three additional chlorpropamide analogues, viz. 4-chloro-N,O-bis(ethoxycarbonyl)-N-hydroxybenzenesulfonamide (NPI-4), N,O-bis(carbomethoxy)methanesulfohydroxamic acid (NPI-5), and 2-[(ethoxycarbonyl)oxy]-1,2-benzisothiazol-3(2H)-one 1,1-dioxide (NPI-6), were evaluated in the present investigation. Quantified were NAD-linked oxidation of benzaldehyde catalyzed by nALDH3A1 and tALDH3A1, and NAD-linked oxidation of acetaldehyde catalyzed by rALDH1A1 and rALDH2, all at 37 degrees C and pH 8.1, and in the presence and absence of inhibitor. NPI-4, NPI-5 and NPI-6 were not substrates for the oxidative reactions catalyzed by any of the ALDHs studied. Oxidative reactions catalyzed by the ALDH3A1s, rALDH1A1 and rALDH2 were each inhibited by NPI-4 and NPI-5. NPI-6 was a poor inhibitor of nALDH3A1- and tALDH3A1-catalyzed oxidations, but was a relatively potent inhibitor of rALDH1A1- and rALDH2-catalyzed oxidations. In all cases, inhibition of ALDH-catalyzed oxidation was directly related to the product of inhibitor concentration and preincubation (enzyme+inhibitor) time. As judged by the product values (microMxmin) required to effect 50% inhibition (IC(50)): (1) nALDH3A1 and tALDH3A1 were essentially equisensitive to inhibition by NPI-4 and NPI-5, and both enzymes were poorly inhibited by NPI-6; (2) rALDH1A1 was, relative to the ALDH3A1s, slightly more sensitive to inhibition by NPI-4 and NPI-5, and far more sensitive to inhibition by NPI-6; and (3) rALDH1A1 was, relative to rALDH2, essentially equisensitive to inhibition by NPI-5, whereas, it was slightly more sensitive to inhibition by NPI-4 and NPI-6.

Effects of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid on extrahepatic sulfhydryl levels in mice treated with acetaminophen.

The cysteine (Cys) precursor 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA) has been shown to protect against acetaminophen (APAP)-induced hepatic GSH, GSSG, and Cys depletion and hepatic necrosis. The aim of this study was to determine the effects of PTCA on the concentrations of sulfhydryl compounds in extrahepatic tissues, including renal cortex, whole blood, and brain, in C57BL/6 mice treated with hepatotoxic doses of APAP. PTCA (1-5 mmol/kg, i.p.) was administered 30 min after the administration of APAP at a dose (800 mg/kg; 5.29 mmol/kg, i.p.) that depleted hepatic GSH and Cys at 4 hr by 95 and 86%, respectively. Tissue concentrations of GSH and Cys were determined by HPLC. At 4 hr following APAP administration, renal cortical GSH and Cys concentrations were decreased to 64 and 39%, respectively, of vehicle-treated control values, and blood concentrations were decreased to 87 and 30%, respectively, of vehicle controls. Brain GSH and Cys were not depleted by APAP. PTCA at 5 mmol/kg (i) attenuated the APAP-induced depletion of GSH and Cys at 4 hr in renal cortex (78 and 65%, respectively, of vehicle controls), (ii) prevented APAP-induced Cys depletion in blood (670% of vehicle controls) with no effect on GSH concentration (94% of vehicle controls), and (iii) increased GSH and Cys concentrations in brain (119 and 411%, respectively, of vehicle controls). The results demonstrate a high degree of tissue selectivity in the APAP-induced depletion of GSH and Cys, and in the effectiveness of PTCA in maintaining and even elevating sulfhydryl levels in extrahepatic tissues of APAP-treated mice.

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.

N-Terminal dipeptides of D(-)-penicillamine as sequestration agents for acetaldehyde.

Since acetaldehyde (AcH), a toxic oxidation product of ethanol, may play an etiologic role in the initiation of alcoholic liver disease, we had earlier pioneered the development of beta, beta-disubstituted-beta-mercapto-alpha-amino acids as AcH-sequestering agents. We now report the synthesis of a series of N-terminal dipeptides of D(-)-penicillamine, prepared from the synthon 3-formyl-2,2,5,5-tetramethylthiazolidine-4S-carboxylic acid (3), a cyclized N-protected derivative of D(-)-penicillamine. These dipeptides were equally or more effective than penicillamine in trapping AcH in a cell-free system. In experiments using a hepatocyte culture system, two of the dipeptides, D-penicillamylglycine (6a) and D-penicillamyl-beta-alanine (6d), at 1/20 the molar concentration of ethanol, lowered the concentration of ethanol-derived AcH by 79% and 84%, respectively, at 2 h. The presence of cyanamide (an inhibitor of aldehyde dehydrogenase) in the incubation medium resulted in a 45-fold increase in ethanol-derived AcH; nevertheless, dipeptides 6a and 6c (D-penicillamyl-alpha-aminoisobutyric acid) were able to reduce this AcH level by approximately one-third.

Reaction of nitroxyl, an aldehyde dehydrogenase inhibitor, with N-acetyl-L-cysteine.

Nitroxyl (HNO) is the aldehyde dehydrogenase (AIDH) inhibitor produced by catalase action on cyanamide. Incubation of N-acetyl-L-cysteine (NAC), a reagent with a free sulfhydryl group, with Piloty's acid (a nitroxyl generator) suggested that NAC was acting as a competitive "trap" for nitroxyl. Elucidation of the structure of this reaction product should give an insight as to how nitroxyl interacts with AIDH, a sulfhydryl enzyme. We now present evidence that the product formed is N-acetyl-L-cysteinesulfinamide (NACS). We have synthesized NACS and showed that this synthetic product was identical to the product formed in the trapping experiment. Both had identical RT values by reverse phase HPLC and identical RF values by TLC using three different solvent systems. The structural identification of this nitroxyl trapped product as a sulfinamide now allows the chemical confirmation of the active-site cysteine residue of AIDH as Cys-302.

Diethylcarbamoylating/nitroxylating agents as dual action inhibitors of aldehyde dehydrogenase: a disulfiram-cyanamide merger.

Benzenesulfohydroxamic acid (Piloty's acid) was functionalized on the hydroxyl group with the N,N-diethylcarbamoyl group, and the hydroxylamine nitrogen was substituted with acetyl (1a), pivaloyl (1b), benzoyl (1c), and ethoxycarbonyl (1d) groups. Only compound 1d inhibited yeast aldehyde dehydrogenase (AlDH) in vitro (IC(50) 169 microM). When administered to rats, 1d significantly raised blood acetaldehyde levels following ethanol challenge, thus serving as a diethylcarbamoylating/nitroxylating, dual action inhibitor of AlDH in vivo. A more potent dual action agent was N-(N, N-diethylcarbamoyl)-O-methylbenzenesulfohydroxamic acid (5c), which was postulated to release diethylcarbamoylnitroxyl (9), a highly potent diethylcarbamoylating/nitroxylating agent, following metabolic O-demethylation in vivo. The dual action inhibition of AlDH exhibited by 1d, and especially 9, constitutes a merger of the mechanism of action of the alcohol deterrent agents, disulfiram and cyanamide.

Generation of nitric oxide and possibly nitroxyl by nitrosation of sulfohydroxamic acids and hydroxamic acids.

Diazeniumdiolates (NONOates) and sulfohydroxamic acids are chemical entities that spontaneously generate nitric oxide (NO) and nitroxyl (HNO), respectively, at physiological pH and temperature. By combining the functional aspects of the NONOates with the hydroxamic acids and sulfohydroxamic acids, hybrid NONOate-type compounds that could theoretically generate nitroxyl or nitric oxide can be rationalized. Although the instability of these compounds, viz., the N-nitrosohydroxamic acids and the N-nitrososulfohydroxamic acids, precluded their chemical characterization by actual isolation, their transient existence was deduced by identification of the products of their decomposition. Thus, treatment of benzohydroxamic acid (BHA) with limiting or excess nitrous acid (from NaNO(2) and H(3)PO(4)) gave rise to quantitative generation of N(2)O, possibly via HNO, based on the limiting reactant. Nitrosation of N-t-butyloxycarbonyl hydroxylamine gave similar results. The organic acid produced from BHA was identified as benzoic acid. No nitric oxide was detected from these reactions. In contrast, treatment of Piloty's acid (benzenesulfohydroxamic acid) and methanesulfohydroxamic acid (MSHA) with nitrous acid under the same conditions as above gave 36% of the theoretical quantity of NO from Piloty's acid and 47% of NO from MSHA, although finite quantities of HNO (measured as N(2)O) were also formed. The organic acid produced from Piloty's acid was identified by reverse-phase HPLC as the redox product, benzenesulfinic acid.

The reaction of nitroxyl (HNO) with nitrosobenzene gives cupferron (N-nitrosophenylhydroxylamine).

Nitroxyl (HNO), a penultimate product in the NOS-catalyzed conversion of L-arginine to L-citrulline, generated from Angeli's salt (AS) was determined by trapping it with nitrosobenzene (NB) to produce cupferron. The cupferron thus produced was characterized by complexation with Fe3+, Al3+, Cu2+, or Sn2+. UV/VIS spectra of the solubilized (in CHCl3) precipitates formed from NB and nitroxyl generated from AS in the presence of the iron, aluminum, copper, or tin salts were identical to those of their corresponding cupferron complexes. The identities of the Fe3+ and Cu2+ complexes formed from NB and HNO were further confirmed by their identical retention times on HPLC when compared to authentic Fe3+ and Cu2+ cupferron complexes. It was possible to detect 5 x 10(-6) M of the cupferron Fe3+ complex spectrophotometrically and to measure its production from the nitroxyl generators AS and methanesulfohydroxamic acid (MSHA) in the presence of 10(-4) M NB. The yield of cupferron was 51 and 62% of the amount of nitroxyl possible from AS or MSHA, respectively, after taking into account the relative rates of nitroxyl generation from these donors.

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 Angeli's salt (Na2N2O3) at physiological pH, and, indeed, Angeli's 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 Angeli's 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).

Prodrugs of nitroxyl and nitrosobenzene as cascade latentiated inhibitors of aldehyde dehydrogenase.

The prototypic aromatic C-nitroso compound, nitrosobenzene (NB), was shown previously to mimic the effect of nitroxyl (HN=O), the putative active metabolite of cyanamide, in inhibiting aldehyde dehydrogenase (AlDH). To minimize the toxicity of NB in vivo, pro-prodrug forms of NB, which were designed to be bioactivated either by an esterase intrinsic to AlDH or the mixed function oxidase enzymes of liver microsomes, were prepared. Accordingly, the prodrug N-benzenesulfonyl-N-phenylhydroxylamine (3) was further latentiated by conversion to its O-acetyl (1a), O-methoxycarbonyl (1b), O-ethoxycarbonyl (1c), and O-methyl (2) derivatives. Similarly, pro-prodrug forms of nitroxyl were prepared by derivatization of the hydroxylamino moiety of methanesulfohydroxamic acid with N, O-bis-acetyl (7a), N,O-bis-methoxycarbonyl (7b), N, O-bis-ethoxycarbonyl (7c), and N-methoxycarbonyl-O-methyl (7d) groups. It was expected that the bioactivation of these prodrugs would initiate a cascade of nonenzymatic reactions leading to the ultimate liberation of NB or nitroxyl, thereby inhibiting AlDH. Indeed, the ester pro-prodrugs of both series were highly active in inhibiting yeast AlDH in vitro with IC50 values ranging from 21 to 64 microM. However, only 7d significantly raised ethanol-derived blood acetaldehyde levels when administered to rats, a reflection of the inhibition of hepatic mitochondrial AlDH-2.

Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry.

S-Nitrosothiols have been implicated to play key roles in a variety of physiological processes. The potential physiological importance of S-nitrosothiols prompted us to examine their reaction with thiols. We find that S-nitrosothiols can react with thiols to generate nitroxyl (HNO) and the corresponding disulfide. Further reaction of HNO with the remaining S-nitrosothiol and thiol results in the generation of other species including NO, sulfinamide, and hydroxylamine. Mechanisms are proposed that rationalize the observed products.

Inhibition of human class 3 aldehyde dehydrogenase, and sensitization of tumor cells that express significant amounts of this enzyme to oxazaphosphorines, by chlorpropamide analogues.

In some cases, acquired as well as constitutive tumor cell resistance to a group of otherwise clinically useful antineoplastic agents collectively referred to as oxazaphosphorines, e.g. cyclophosphamide and mafosfamide, can be accounted for by relatively elevated cellular levels of an enzyme, viz. cytosolic class 3 aldehyde dehydrogenase (ALDH-3), that catalyzes their detoxification. Ergo, inhibitors of ALDH-3 could be of clinical value since their inclusion in the therapeutic protocol would be expected to sensitize such cells to these agents. Identified in the present investigation were two chlorpropamide analogues showing promise in that regard, viz. (acetyloxy)[(4-chlorophenyl)sulfonyl]carbamic acid 1,1-dimethylethyl ester (NPI-2) and 4-chloro-N-methoxy-N-[(propylamino)carbonyl]benzenesulfonamide (API-2). Each inhibited NAD-linked benzaldehyde oxidation catalyzed by ALDH-3s purified from human breast adenocarcinoma MCF-7/0/CAT cells (IC50 values were 16 and 0.75 microM, respectively) and human normal stomach mucosa (IC50 values were 202 and 5 microM, respectively). The differential sensitivities of stomach mucosa ALDH-3 and breast tumor ALDH-3 to each of the two inhibitors can be viewed as further evidence that the latter is a subtle variant of the former. Human class 1 (ALDH-1) and class 2 (ALDH-2) aldehyde dehydrogenases were much less sensitive to NPI-2; IC50 values were >300 microM in each case. API-2, however, did not exhibit a similar degree of specificity; IC50 values for ALDH-1 and ALDH-2 were 7.5 and 0.08 microM, respectively. Each sensitized MCF-7/0/CAT cells to mafosfamide; the LC90 value decreased from >2 mM to 175 and 200 microM, respectively. Thus, the therapeutic potential of combining NPI-2 or API-2 with oxazaphosphorines is established.

Novel prodrugs of cyanamide that inhibit aldehyde dehydrogenase in vivo.

S-Methylisothiourea (4), when administered to rats followed by a subsequent dose of ethanol, gave rise to a 119-fold increase in ethanol-derived blood acetaldehyde (AcH) levels-a consequence of the inhibition of hepatic aldehyde dehydrogenase (A1DH)-when compared to control animals not receiving 4. The corresponding O-methylisourea was totally inactive under the same conditions, suggesting that differential metabolism may be a factor in this dramatic difference between the pharmacological effects of O-methylisourea and 4 in vivo. The S-n-butyl- and S-isobutylisothioureas (8 and 9, respectively) at doses equimolar to that of 4 were nearly twice as effective in raising ethanol-derived blood AcH, while S-allylisothiourea (10) was slightly less active. However, blood ethanol levels of all experimental groups were indistinguishable from controls. Pretreatment of the animals with 1-benzylimidazole, a known inhibitor of the hepatic mixed function oxidases, followed sequentially by either 8, 9, or 10 plus ethanol, reduced blood AcH levels by 66-88%, suggesting that the latter compounds were being oxidatively metabolized to a common product that led to the inhibition of AcH metabolism. In support of this, when 8 was incubated in vitro with rat liver microsomes coupled to catalase and yeast A1DH, the requirement for microsomal activation for the inhibition of A1DH activity was clearly indicated. We suggest that S-oxidation is involved and that the S-oxides produced in vivo inhibited A1DH directly, or spontaneously released cyanamide, an inhibitor of A1DH. Indeed, incubation of 8 with rat liver microsomes and NADPH gave rise to cyanamide as metabolite, identified as its dansylated derivative. Cyanamide formation was minimal in the absence of coenzyme. Extending the side chain was detrimental, since S-benzylisothiourea (11) and S-n-hexadecylisothiourea (12) were toxic, the latter producing extensive necrosis of the liver and kidneys when administered to rats.

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 (Ellman's 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.

Nitrosyl cyanide, a putative metabolic oxidation product of the alcohol-deterrent agent cyanamide.

When incubated with catalase/glucose-glucose oxidase, 13C-labeled cyanamide gave rise not only to 13C-labeled cyanide, but also to 13C-labeled CO2. Moreover, a time-dependent formation of nitrite was observed when cyanamide was oxidized in this system. These results suggested that the initial product of cyanamide oxidation, viz. N-hydroxycyanamide, was being further oxidized by catalase/H2O2 to nitrosyl cyanide (O = N-C = N). Theoretically, nitrosyl cyanide can hydrolyze to the four end-products detected in the oxidative metabolism of cyanamide in vitro, viz. nitroxyl, cyanide, nitrite, and CO2. Accordingly, both unlabeled and 13C-labeled nitrosyl cyanide were synthesized by the low temperature (-40 to -50 degrees) nitrosylation of K-(18-crown-6)cyanide with nitrosyl tetrafluoroborate. The product, a faint blue liquid at this temperature, was transferred as a gas to phosphate-buffered solution, pH 7.4, where it was solvolyzed. Analysis of the headspace by gas chromatography showed the presence of N2O, the dimerization/dehydration product of nitroxyl, while cyanide was detected in the aqueous solution, as measured colorimetrically. [13C]CO2 was analyzed by GC/MS. An oxidative biotransformation pathway for cyanamide that accounts for all the products detected and involving both N-hydroxycyanamide and nitrosyl cyanide as tandem intermediates is proposed.