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PubMed2001

In vivo inhibition of aldehyde dehydrogenase by disulfiram.

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not yet been determined. In this report, we demonstrate a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (rmALDH) were purified in one-step with an affinity cartridge. The in vivo DSF-treated rmALDH showed 77% inhibition in enzyme activity as compared to that of the control. Subsequently, the control and DSF-inhibited rmALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited rmALDH as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+)=973.7 and 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO).

Aldehyde Dehydrogenase↗
PubMed2001

Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.

Disulfiram (Ds), a clinically employed alcohol deterrent of the thiuram disulfide (TD) class of compounds, is known to cause hepatitis and neuropathies. Although this drug has been shown to inhibit different thiol-containing enzymes, the actual mechanism of Ds toxicity is not clear. We have previously demonstrated that Ds impairs the permeability of inner mitochondrial membrane (IMM) [Arch. Biochem. Biophys. 356 (1998) 46]. In this report, the effect of Ds and its structural analogue thiram (Th) on mitochondrial functions was studied in detail. We found that mitochondria metabolize TDs in a NAD(P)H- and GSH-dependent manner. At the concentration above characteristic threshold, TDs induced irreversible oxidation of NAD(P)H and glutathione (GSH) pools, collapse of transmembrane potential, and inhibition of oxidative phosphorylation. The presence of Ca(2+) and exhaustion of mitochondrial glutathione (GSH+GSSG) decreased the threshold concentration of TDs. Swelling of the mitochondria and leakage of non-transported fluorescent dye BCECF from the matrix indicated that TDs induced the mitochondrial permeability transition (MPT). Mitochondrial permeabilization by TDs involves two, apparently distinct mechanisms. In the presence of Ca(2+), TDs produced cylosporin A-sensitive swelling of mitochondria, which was inhibited by ADP and accelerated by carboxyatractyloside (CATR) and phosphate. In contrast, the swelling produced by TDs in the absence of Ca(2+) was not sensitive to cyclosporin A (CsA), ADP and CATR but was inhibited by phosphate. Titration with N-ethylmaleimide revealed that these two mechanisms involve different SH-groups and probably different transport proteins on the IMM. Our findings indicate that at pharmacologically relevant concentrations TDs may cause an irreversible mitochondrial injury as a result of induction of the MPT.

Adenosine Diphosphate↗
PubMed2003

Inactivation of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L. leaves by disulfiram.

Betaine aldehyde dehydrogenase (BADH) activity might be crucial for the growth of the human pathogen Pseudomonas aeruginosa under conditions of infection and therefore appears to be a suitable target for antimicrobial agents. As a first step in the search for BADH inhibitors, we have tested the effects of the known aldehyde dehydrogenase inhibitor disulfiram (DSF) on the activity of P. aeruginosa and Amaranthus hypochondriacus (amaranth) leaf BADHs. DSF totally inactivated both enzymes in a time- and dose-dependent manner. In the case of the Pseudomonas enzyme, inactivation kinetics were monophasic with a second-order inactivation rate constant at pH 6.9 of 4.9+/-0.4 M(-1) s(-1), whereas the plant enzyme was inactivated in a biphasic process with second-order inactivation rate constants at pH 7.5 of 6.8+/-0.6 and 0.33+/-0.04 M(-1) s(-1). At pH 8.8, the second-order rate constants for inactivation of the bacterial enzyme was 1 x 10(3) M(-1) s(-1), which compare well with that reported for human liver mitochondrial aldehyde dehydrogenase (ALDH2), the target of DSF inhibition in the aversion therapy of alcoholism. Both BADHs were inactivated faster in the presence of NAD(P)(+) than in its absence, whereas NAD(P)H and betaine aldehyde protected the bacterial, but increased the inactivation rate of the plant enzyme. The inactivated enzymes were reactivated by dithiothreitol, but not by a high concentration of the physiological reductant glutathione. The high in vitro sensitivity of the Pseudomonas BADH to DSF, particularly in the presence of NAD(P)(+), together with the lack of reversibility of DSF modification by glutathione, makes this inhibitor a potential antimicrobial agent and suggests that it might be worth testing its effects and those of its metabolites in vivo, under culture conditions in which the activity of BADH is required for growth of the bacteria.

Aldehyde Oxidoreductases↗
PubMed1996

Mixed disulfides from disulfiram inhibit the benzo[a]pyrene induced mutagenesis.

Some mixed disulfides derived from disulfiram and endogenous thiol compounds have been synthesized, biochemically characterized and their potential antigenotoxic effects have been proposed. The present study evaluated the mutagenic and antimutagenic specificities of two mixed disulfides using S. typhimurium reversion assay, namely S-(N,N-diethyldithiocarbamoyl)-N-acetylcysteine (AC-DDTC) and-L-glutathione (GS-DDTC). The two mixed disulfides were not mutagenic to Salmonella strains TA98 and TA100 in the presence or absence of S9 mixture. The increased number of revertants by benzo[a]pyrene (B(a)P) has been reduced to the control level by the preincubation with AC-DDTC or GS-DDTC. It was not due to the killing effect of B(a)P, mixed disulfides or B(a)P-disulfide mixture. The antimutagenic effect of AC-DDTC was more potent than that of GS-DDTC. These results indicate that AC-DDTC and GS-DDTC may have a role to play in reducing the risk of mutagenic effects of B(a)P.

Animals↗
PubMed1983

Delirium with phenytoin and disulfiram administration.

The case of a 54-year-old man with delirium secondary to phenytoin and disulfiram administration is presented. The pharmacology, interaction, and resulting toxicity of these two drugs are explored. The patient made an uneventful recovery when the medications were withheld.

Delirium↗
PubMed1999

Sister-chromatid exchanges induced by disulfiram in bone marrow and spermatogonial cells of mice treated in vivo.

Disulfiram is a widely used drug to treat alcoholism due to its capacity to inhibit the metabolism of acetaldehyde; however, its genotoxic potential is not well known. Thus, the aim of this investigation was to determine whether the chemical may induce sister-chromatid exchanges (SCEs) in an in vivo study using mouse bone marrow and spermatogonial cells. We used doses of 200, 400 and 800 mg/kg body weight and compared the obtained data with the values determined in a negative control group as well as with a positive control group (cyclophosphamide, 50 mg/kg). The results in both systems indicated a weak genotoxic response by the chemical. In the case of bone marrow, a significant SCE level was achieved only with the high tested dose, but in spermatogonial cells the three doses tested showed a significant difference with respect to the negative control. No significant alterations in the mitotic index or in the cell proliferation kinetics were observed in somatic cells. Concerning the effect of cyclophosphamide, an increase in the level of SCEs was observed in both types of cells, reaching more than three times the values obtained in their respective control groups.

Alcohol Deterrents↗
PubMed1997

Disulfiram is a potent inhibitor of proteases of the caspase family.

We have recently shown that dithiocarbamate (DC) disulfides inhibit proteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocytes treated with anti-CD95 (Fas/APO-1) antibody. Because the processing can be accomplished by caspase activity, we investigated the effect of DC disulfides, such as disulfiram (DSF), on active caspases. DSF showed a dose-dependent inhibition was prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Direct interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopain). An apparent rate constant (K(app)) for this inhibition was estimated to be 0.45 x 10(3)M(-1)s(-1). DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-converting enzyme, ICE), with a K(app) of 2.2 x 10(3) M(-1)s(-1). In this case protein mixed disulfide formation between DSF and caspase-1 was directly demonstrated using 35S-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A glutathione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that caspases are quite sensitive to thiol oxidation and that DSF is a very potent oxidant of caspase protein thiol(s), being 700-fold more potent than glutathione disulfide.

Alcohol Deterrents↗
PubMed2000

Disulfiram generates a stable N,N-diethylcarbamoyl adduct on Cys-125 of rat hemoglobin beta-chains in vivo.

Disulfiram (DSF) is a drug used in aversion therapy to treat alcoholics and acts by inhibiting mitochondrial low-K(m) aldehyde dehydrogenase. Investigations into the mechanisms for in vivo inactivation suggest that the DSF metabolite S-methyl-N, N-diethylthiocarbamate sulfoxide reacts irreversibly with an active site Cys. This work aimed to determine if DSF generates monothiocarbamate adducts on cysteine residues in vivo by examining hemoglobin. Sprague-Dawley rats were treated with DSF po for 2, 4, and 6 weeks. Rats have four different globin beta-chains, of which three (beta-1-3) contain two cysteine residues each. MALDI-TOF MS analysis of two new globin species from DSF-treated rats collected by HPLC revealed increments of 99 Da above the mass of the unmodified chains (beta-2 and beta-3). In a separate experiment, the globin mixture was digested for 2 h with Glu-C and reanalyzed by MALDI-TOF MS. Results showed a peptide at m/z 2716.3 having a mass 99 Da higher than a known Cys-containing peptide. Subsequently, the Glu-C digest was analyzed using Q-TOF tandem MS, enabling observation of the +4 charge state of the peptide with m/z 2716.3. This peptide was fragmented to produce y-sequence ions that located the modification to Cys-125 (present on both beta-2 and beta-3). Cys-125 is the most reactive of two cysteine residues on these beta-chains. To confirm the structure of the modification, globin was hydrolyzed with 6 N HCl at 110 degrees C for 18 h. The adduct survived these conditions so that S-(N,N-diethylcarbamoyl)cysteine was detected in the hydrolysates of treated rats on the basis of comparison with the tandem MS spectrum of a standard. These results extend the findings of others obtained using glutathione conjugates and demonstrate the ability of DSF to covalently modify Cys residues of proteins in a manner consistent with the production of S-methyl-N, N-diethylthiocarbamate sulfoxide, or sulfone, intermediates.

Alcohol Deterrents↗
PubMed1976

The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver.

The effect of some close structural analogues of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. Several thiuram disulphides are equally potent inhibitors of the cytoplasmic enzyme, but in all cases the mitochondrial enzyme is much less strongly affected. Tetramethylthiuram monosulphide decreases the activity of the cytoplasmic enzyme in a process apparently involving a covalent interaction.

Aldehyde Oxidoreductases↗
PubMed1979

2,2'-Dithiodipyridine activates aldehyde dehydrogenase and protects the enzyme against inactivation by disulfiram.

Small concentrations of 2,2'-dithiodipyridine cause a rapid activation of sheep liver cytoplasmic aldehyde dehydrogenase in the presence of NAD+. Enzyme pre-modified by 2,2'-dithiodipyridine is largely protected against the potent inactivatory effect of disulfiram. 2,2'-Dithiobis-(5-nitropyridine) inactivates the enzyme. The implications of these results are discussed with reference to various possible classes of thiol group in aldehyde dehydrogenase.

2,2'-Dipyridyl↗
PubMed1997

Comparative effects of disulfiram and diethyldithiocarbamate against testicular toxicity in rats caused by acute exposure to cadmium.

Disulfiram (DSF) and diethyldithiocarbamate (DED) were compared for their protective effects against the testicular toxicity induced by acute exposure to cadmium (Cd) in rats. Rats were injected subcutaneously with CdCl2 126.7 mumol (3 mg) Cd/kgl, and 30 min later they were injected intraperitoneally with DSF (0.05-0.5 mmol/kg) or DED (0.1-1 mmol/kg). The treatment with DSF at dose levels of 0.1-0.5 mmol/kg prevented the increases in testicular lipid peroxidation and calcium (Ca) concentrations and the decreases in testicular weight that were observed at 7 d after Cd injection. DED at dosage levels of 0.2-1 mmol/kg likewise reduced Cd-induced testicular toxicity. An increase in testicular iron (Fe) concentrations at 7 d and sterility at 59 d after Cd injection were almost completely blocked by treatment with DSF or DED at the highest doses, but lower doses of DSF or DED were ineffective. These results indicated that DSF, which is metabolized to DED, had a protective effect against Cd-induced testicular toxicity nearly equivalent to DED at approximately one-half the dose.

Animals↗
PubMed2004

Bioavailability and anticataract effects of a topical ocular drug delivery system containing disulfiram and hydroxypropyl-beta-cyclodextrin on selenite-treated rats.

PURPOSE: To test the effect of aqueous eye drops containing a high concentration of disulfiram (DSF) in a cyclodextrin- based drug delivery system. This system increases both the drug solubility in aqueous eye drops and the permeability of drug into the rabbit eye, by the formation of a drug-cyclodextrin inclusion complex, and so enhances the ocular bioavailability and anti-cataract effect of DSF. METHODS: The DSF and hydroxypropyl-beta-cyclodextrin (HPbetaCD) inclusion (DSF/HPbetaCD) was studied using solubility methods, IR spectra and X-ray diffraction patterns. Suitable formulations for DSF eye drops were first identified by a trans-corneal penetration experiment in vitro. Finding a new p-bromophenacyl bromide (p-BPB) derivative reagent for diethyldithiocarbamic acid (DDC), which was a metabolite of DSF, allowed precise determination of the contents of DSF in aqueous humor. The ocular bioavailability was calculated by a transcorneal experiment of DSF in vivo. The lens opacity of a selenite-induced cataract in rat pups was monitored using a slit lamp with an anterior eye segment analysis system. RESULTS: The formation of DSF/HPbetaCD inclusion and the addition of hydroxypropylmethylcellulose (HPMC), as a penetration enhancer, played very important roles in increasing the ocular bioavailability of DSF. DSF eye drops, with a formulation of 1.26% (w/v) DSF/HPbetaCD inclusion, 0.01% (w/v) HPMC, 0.005% (w/v) benzalkonium chloride and 0.9% (w/v) sodium chloride, inhibited the onset of selenite-induced cataracts effectively. CONCLUSIONS: The cyclodextrin-based drug delivery system enhances both the solubility of DSF in aqueous eye drops and permeability of the drug into the rabbit eye. DSF ocular bioavailability in rabbit aqueous humor exceeded those reported for the DSF ophthalmic preparation. DSF eye drops effectively prevent the development of selenite-induced cataracts.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed2003

Learning the language of abstinence in addiction treatment: some similarities between relapse-prevention with disulfiram, naltrexone, and other pharmacological antagonists and intensive "immersion" methods of foreign language teaching.

Relapse-prevention (RP) is an educational process. Learning to abstain from alcohol or opiates after years of dependence involves selectively suppressing old, maladaptive habits of thought and behavior and establishing new, adaptive ones. This process resembles foreign language (FL) learning. Effective FL teaching techniques are relevant to RP. "Immersion," the most effective FL teaching method, discourages students from using their first language ab initio, requiring them to use the FL instead, however inexpertly. It resembles exposure and response-prevention for phobic or compulsive disorders. Supervised disulfiram aids RP by discouraging alcoholics from responding to real-life drinking cues in the "language" of excessive drinking, requiring them, ab initio, to practice new, alcohol-free responses. Supervised or depot naltrexone acts similarly in opiate dependence. We discuss the concept of antagonist-assisted abstinence.

Adaptation, Psychological↗
PubMed1988

Urinary thioether biological monitoring in the interaction between 1,2-dichloroethane and disulfiram in Sprague-Dawley rats.

The interaction between inhaled 1,2-dichloroethane (ethylene dichloride; EDC) and dietary Disulfiram (tetraethylthiuram disulfide; Antabuse; DSF) was investigated for male Sprague-Dawley rats in terms of urinary levels of thio-compounds extractable in ethyl acetate and then hydrolyzed in alkali (the classic urinary thioether assay). The assay was found to be an inadequate biological monitoring indicator for EDC or DSF exposure during the DSF/EDC interaction at exposures of 0, 153, 304 and 455 ppm EDC (7 hr/day, 5 days/week, 30 exposure days) for rats fed with AIN-76 diet fortified with 0.15% DSF. EDC inhibited the excretion of DSF-derived thio-compounds with increasing EDC concentration; the thioether content was dose-related in the absence of DSF. In situations where confounding agents generate neutral S-containing urinary metabolites without involvement of endogenous glutathione, the classic thioether assay requires supplementation by other biochemical monitoring strategies.

Administration, Inhalation↗
PubMed2005

An open randomized study comparing disulfiram and acamprosate in the treatment of alcohol dependence.

AIMS: To compare the efficacy of acamprosate (ACP) and disulfiram (DSF) for preventing alcoholic relapse in routine clinical practice. METHODS: One hundred alcoholic men with family members who would encourage medication compliance and accompany them for follow-up were randomly allocated to 8 months of treatment with DSF or ACP. Weekly group psychotherapy was also available. The psychiatrist, patient, and family member were aware of the treatment prescribed. Alcohol consumption, craving, and adverse events were recorded weekly for 3 months and then fortnightly. Serum gamma glutamyl transferase was measured at the start and the end of the study. RESULTS: At the end of the trial, 93 patients were still in contact. Relapse (the consumption of >5 drinks/40 g of alcohol) occurred at a mean of 123 days with DSF compared to 71 days with ACP (P = 0.0001). Eighty-eight per cent of patients on DSF remained abstinent compared to 46% with ACP (P = 0.0002). However, patients allocated to ACP had lower craving than those on DSF (P = 0.002). CONCLUSION: DSF is superior to ACP for preventing relapse in alcohol-dependent men with good family support. Further comparisons between these two drugs in different treatment settings and populations are warranted.

Acamprosate↗
PubMed1991

Effect of disulfiram on 1,2-dimethylhydrazine- and azoxymethane-induced aberrant crypt foci.

We have previously reported a method for visualizing the mucosal surface of fixed unsectioned rodent colons at the crypt level and have identified lesions, termed aberrant crypt foci (ACF), in the colons of carcinogen-treated rodents. We hypothesized that ACF represent the precursor lesions (PL) of colon cancer. In the present study, the effect of feeding disulfiram (DSF) added to a semi-synthetic diet (0.5% or 1% by wt) on 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM) induced ACF was investigated. DSF has been shown to inhibit DMH and AOM-induced colon cancer. Therefore, it was reasoned that if ACF represent PL then their induction and growth should also be inhibited by DSF. CF1 female mice were randomly divided into three groups of 30 each. Group 1 was fed a diet containing 1% DSF for 9 days prior to and 1 day after receiving a single i.p. injection of either DMH, AOM or saline. Group 2 was fed a diet containing 1% DSF for 9 days prior to and 14 days after receiving a single i.p. injection of DMH, AOM or saline, whereas group 3 received control diet throughout the experimental duration. All animals were killed 5 weeks after receiving the injections. It was observed that feeding DSF, for 9 days prior to and for either 1 day or 14 days after the administration of a single injection of DMH, resulted in a complete inhibition of ACF. DSF feeding for 9 days prior to and 1 day after AOM injection resulted in a significantly greater number of ACF compared to the control group (12 +/- 2.3 vs 7.2 +/- 1.2); whereas DSF feeding for a longer duration (i.e. 9 days prior to and 14 days after AOM treatment) was associated with a significantly lower number of ACF compared to those fed DSF for only one day after AOM treatment (4.1 +/- 0.6 vs 12.4 +/- 2.3) and a lower number compared to the control group (4.1 +/- 0.6 vs 7.2 +/- 1.2).

1,2-Dimethylhydrazine↗
PubMed1982

In vitro formation of methyldiethyldithiocarbamate after the reaction of nitrosoacetoxymethylmethylamine or methylnitrosourea with disulfiram.

Analysis by scintillation measurement, mass spectrometry and h.p.l.c. showed that diethyldithiocarbamic acid (DDTC), the main metabolite of disulfiram (DSF), forms methyldithiocarbamate (MeDDTC) when incubated with [14C]nitrosoacetoxymethylmethylamine ([14C]NAMM) or [14C]methylnitrosourea ([14C]MNU) in different media (bacteria, esterases, rat liver 9000 x g supernatant fraction and microsomes). When DSF instead of DDTC was used, MeDDTC was formed only when soluble enzymes were present which are required to split DSF into two DDTC moieties. No physiological methylation of DDTC takes place as was shown by experiments with [3H]MNU. [14C]Methanol, formed by the decay of [14C]NAMM and [14C]MNU was shown to have no alkylating properties.

Animals↗
PubMed1984

Chemical interaction of disulfiram with nitrosodimethylamine after in vitro enzymatic activation.

The in vitro reaction between disulfiram (DSF) and N-nitroso[14C]dimethylamine [( 14C]NDMA) was studied. Incubations of DSF with [14C]NDMA were carried out in the presence of rat liver microsomes, control 9000 g (S9) supernatant fraction and phenobarbital-induced S9 fraction. H.p.l.c. analysis and liquid scintillation measurement provided evidence for the formation of methyldiethyldithiocarbamate (MeDDTC) as a product of the reaction between diethyldithiocarbamate (DDTC), the main active metabolite of DSF and the 'methyl-cation' released by NDMA after enzymatic activation. The amount of MeDDTC found here was consistent with the rate of oxidation of NDMA to formaldehyde. Scintillation counting confirmed that other radioactive peaks, not due to MeDDTC, were unrelated to the methylation of L-cysteine by [14C]NDMA.

Animals↗