Inhibition Of ALDH Research Articles

Abstract 488: New therapeutic strategies suppressing NAD+ and ATP production in cancer metabolism specific to 3D cultured ovarian cancer cells

Abstract Ovarian cancer is the gynecological cancer with the highest mortality rate. As resistance to treatment is acquired and many patients relapse, new treatment strategies are needed. A model that strongly reflects the features of recurrent ovarian cancer in the peritoneal cavity is the "cancer stem cells (CSCs)," which are enriched when cells are cultured in three dimensions (3D) on low-attachment plates. The unique metabolic system of the "3D CSCs" is the subject of a novel therapeutic approach. Here, we show that ovarian cancer 3D CSCs have strong NAD+ production capacity, which is particularly dependent on nicotinamide phosphoribosyl transferase (NAMPT), known as the rate-limiting enzyme in the salvage pathway of NAD+ production. We found that NAMPT inhibition blocks NADPH and ATP production as well as NAD+, and subsequently suppresses the synthesis capacity of proteins, fat droplets, and cholesterol in the 3D ovarian cancer cells. In addition, we found that the combination of NAMPT inhibitor and the ALDH inhibitor disulfiram had a pronounced synergistic effect. This synergy inhibits oxidative phosphorylation and ALDH activity and promotes reactive oxygen species and apoptosis via caspase 3/7 cleavage, which leads to death of the 3D cultured cells. Our results demonstrate that high NAMPT expression in ovarian cancer is likely to be a strong poor prognostic factor and that the combination of a NAMPT inhibitor and disulfiram could be a new treatment option to treat or prevent recurrent ovarian cancer. Our novel findings focus on NAMPT addiction as a property of ovarian cancer 3D CSCs, and strongly suggest the combined inhibition of NAMPT and ALDH as a potential therapeutic strategy. Citation Format: Kei Kudo, Brittney S. Harrington, Franklin Ning, Rahul Kamdar, Yusuke Shibuya, Soumya Korrapati, Maria Fergusson, Calen Kucera, Fantini Massimo, Christina M. Annunziata. New therapeutic strategies suppressing NAD+ and ATP production in cancer metabolism specific to 3D cultured ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 488.

Anti-Proliferation Effect of Theasaponin E₁ on the ALDH-Positive Ovarian Cancer Stem-Like Cells.

Ovarian cancer has the highest mortality rate of all gynecological malignancies and the five-year death rate of patients has remained high in the past five decades. Recently, with the rise of cancer stem cells (CSCs) theory, an increasing amount of research has suggested that CSCs give rise to tumor recurrence and metastasis. Theasaponin E1 (TSE1), which was isolated from green tea (Camellia sinensis) seeds, has been proposed to be an effective compound for tumor treatment. However, studies on whether TSE1 takes effect through CSCs have rarely been reported. In this paper, ALDH-positive (ALDH+) ovarian cancer stem-like cells from two platinum-resistant ovarian cancer cell lines A2780/CP70 and OVCAR-3 were used to study the anti-proliferation effect of TSE1 on CSCs. The ALDH+ cells showed significantly stronger sphere forming vitality and stronger cell migration capability. In addition, the stemness marker proteins CD44, Oct-4, Nanog, as well as Bcl-2 and MMP-9 expression levels of ALDH+ cells were upregulated compared with the original tumor cells, indicating that they have certain stem cell characteristics. At the same time, the results showed that TSE1 could inhibit cell proliferation and suspension sphere formation in ALDH+ cells. Our data suggests that TSE1 as a natural compound has the potential to reduce human ovarian cancer mortality. However, more research is still needed to find out the molecular mechanism of TSE1-mediated inhibition of ALDH+ cells and possible drug applications on the disease.

P08.26 ATP depletion induced by dual inhibition of ALDH and mitochondrial complex I make therapeutic response in glioblastoma

P08.26 ATP depletion induced by dual inhibition of ALDH and mitochondrial complex I make therapeutic response in glioblastoma

Aldehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity

IntroductionHigh expression of aldehyde dehydrogenase1A1 (ALDH1A1) is observed in many organs and tumors and may identify benign and cancer stem cell populations.MethodsIn the current study, the stem cell characteristics were determined in cells isolated from human prostate cell lines and clinical prostate specimens based upon the ALDEFLUOR™ assay. Cells isolated based on the ALDEFLUOR™ assay were compared to cells isolated based on ATP binding cassette transporter G2 (ABCG2) activity using the side population assay. To test for stem cell characteristics of self-renewal and multipotency, cells with high and low ALDH1A1 activity, based on the ALDEFLUOR™ assay (ALDHHi and ALDHLow), were isolated from prostate clinical specimens and were recombined with rat urogenital sinus mesenchyme to induce prostate gland formation.ResultsThe percentage of ALDHHi cells in prostate cell lines (RWPE-1, RWPE-2, CWR-R1, and DU-145) was 0.5 to 6%, similarly in non-tumor and tumor clinical specimens the percentage of ALDHHi cells was 0.6 to 4%. Recombinants using ALDHHi cells serially generated prostate tissue up to three generations with as few as 250 starting cells. Immunohistochemical analysis of the recombinants using ALDHHi cells contained prostatic glands frequently expressing androgen receptor (AR), p63, chromogranin A, ALDH1A1, ABCG2, and prostate specific antigen (PSA), compared to their ALDHLow counterparts. Inhibition of ALDH resulted in the reduction of sphere formation capabilities in the CWR-R1, but not in the RWPE-2 and DU-145, prostate cell lines. ABCG2 inhibition resulted in a more robust decrease of sphere formation in androgen sensitive cell lines, CWR-R1 and RWPE-2, but not androgen insensitive DU-145. ALDH1A1 expression was enriched in ALDHHi cells and non-side population cells. ABCG2 expression was only enriched in side population cells.ConclusionsThe percentage of ALDHHi cells in prostate cell lines and prostate tissue was consistently higher compared to cells with high ABCG2 activity, identified with the side population assay. The expression of the stem and differentiation markers indicates the ALDHHi recombinants contained cells with self-renewal and multipotency activity. When the two assays were directly compared, cells with the side population phenotype demonstrated more stem cell potential in the tissue recombination assay compared to ALDHHi cells. The increased stem cell potential of side population cells in the tissue recombination assay and the decrease in sphere formation when ABCG2 is inhibited indicates that the side population enriches for prostate stem cells.

Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells

Osteosarcoma (OS) is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR) pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker) activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) gene expression and inhibits K7M2 proliferation, migration, and invasion in vitro. Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis.

A DEAB-sensitive aldehyde dehydrogenase regulates hematopoietic stem and progenitor cells development during primitive hematopoiesis in zebrafish embryos

Although aldehyde dehydrogenase (ALDH) activity has become a surrogate of hematopoietic stem and progenitor cells (HSPCs), its function during hematopoiesis was unclear. Here, we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. Zebrafish embryos were treated with diethylaminobenzaldehyde (DEAB, 1 μmol/l) between 0- and 48 hour-post-fertilization (hpf). MOs targeting aldhs were injected between 1 and 4-cell stage. The effects on hematopoiesis were evaluated at different stages. DEAB treatment between 0 and 18 hpf increased gene expression associated with HSPC (scl, lmo2), erythropoiesis (gata1, α- and β-eHb) and myelopoiesis (spi1) as well as gfp(+) cells in dissociated Tg(gata1:gfp) embryos. The effects were ameliorated by all-trans retinoic acid (1 nmol/l). Definitive hematopoiesis and the erythromyeloid precursors were unaffected. In all, 14 out of 15 zebrafish aldhs were detectable by reverse transcription PCR in 18 hpf embryos, of which only aldh1a2 and aldh16a1 were expressed in sites pertinent to hematopoiesis. Molecular targeting by MOs was demonstrated for 15 aldhs, but none of them, even in combined aldh1a2 and aldh1a3 knockdown, recapitulated the hematopoietic expansion in DEAB-treated embryos. In conclusion, DEAB expands HSPC population during primitive hematopoiesis through inhibition of aldh and retinoic acid synthesis. The specific aldh isoform(s) remains to be determined.

Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

Salivary aldehyde dehydrogenase—Reversible oxidation of the enzyme and its inhibition by caffeine, investigated using fluorimetric method

Objective We have applied fluorimetric method to monitor aldehyde dehydrogenase (ALDH*) activity in human saliva samples to study inactivation, reactivation and inhibition of the enzyme. Design Saliva samples were collected to buffer stock solution, containing various thiols, and assayed in the presence of the fluorogenic substrate 6-dimethylamino-2-naphthaldehyde and NAD +. Fluorescence of the produced 6-dimethylamino-2-naphthalene carboxylate was used to measure the reaction rate. Results Kinetic parameters for the highly fluorogenic substrate, 6-dimethylamino-2-naphthaldehyde were measured, with apparent K m of 7.9 μM at pH 7.3. The apparent K m for NAD + was 1.2 μM. The observed ALDH activity is unstable in the absence of thiols, but can be stabilized by 1 mM glutathione, and inactivated enzyme can be re-activated within 10 min by treatment of 0.5 mM DTT. Two-assay procedure was applied to measure degree of inactivation of ALDH in saliva samples. It was found that degree of ALDH inactivation in fresh samples, stabilized by glutathione, is between 0% and 90%, with average value ca. 40%. Caffeine and theophylline were shown to be moderate inhibitors of salivary ALDH. Conclusions Oxidation of the salivary ALDH in fresh saliva may be reliably measured using fluorimetric two-assay procedure. Preliminary statistics indicate that in most individuals this enzyme is partially inactive. Inhibition of the salivary ALDH by caffeine may have consequences for nutrition safety.

Modulation of Aldehyde Dehydrogenase and Retinoid Signaling Induces the Expansion of Human Hematopoietic Stem Cells.

Aldehyde dehydrogenases are cytosolic enzymes that convert aldehydes into carboxylic acids. Human aldehyde dehydrogenase 1 (ALDH1) is highly expressed in the liver and in hematopoietic stem cells (HSCs). Although ALDH1 is a selectable marker of HSCs, its HSC-specific function is unknown. We hypothesized that ALDH might play a critical role in HSC fate determinations since it is required for the production of retinoic acids, which are broadly implicated in tissue differentiation, tissue patterning and embryonic development in vertebrates. In this study, highly purified human CD34+CD38−lin− HSCs were cultivated with early acting cytokines, thrombopoietin, stem cell factor and Flt-3 ligand (TSF) in the presence or absence of the competitive ALDH inhibitor, diethylaminobenzaldehyde (DEAB). Treatment of human BM and CB HSCs with TSF x 7 days caused a loss of CD34+CD38− cells in culture, morphologic differentiation, amplification of committed colony forming cells (CFCs) and a loss of primitive cells capable of repopulating non-obese diabetic/severe combined immunedeficient mice (SCID-Repopulating Cells, SRCs). Conversely, 7 day culture of BM and CB HSCs with TSF plus 100 μM DEAB blocked the morphologic differentiation and lineage commitment of HSCs in culture, expanded the CD34+CD38− population, and amplified SRCs 2-fold compared to input, indicating a fundamental role for ALDH in HSC differentiation. The effects of DEAB could be reversed by the co-administration of the retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), suggesting that the ability of ALDH to produce retinoic acids is important in determining HSC fate. Via screening studies of direct ligands of RAR and RXR, we also identified a selective RXR modulator which functioned similarly to DEAB by impeding HSC differentiation and causing the 4-fold expansion of SRCs. Interestingly, treatment with either DEAB or the RXR modulator reversed the down-modulation of HOXB4 transcription that was otherwise observed in CD34+CD38−lin- cells during culture with cytokines, suggesting that inhibition of ALDH or RXR modulation may promote HSC self-renewal via discrete interactions with other regulatory pathways. Modulation of ALDH activity and retinoid signaling is a novel and effective strategy to amplify human HSCs.

Modulating carbonyl cytotoxicity in intact rat hepatocytes by inhibiting carbonyl metabolizing enzymes. II. Aromatic aldehydes

The molecular cytotoxic mechanisms of dietary benzaldehydes towards hepatocytes and its modulation by metabolizing enzymes were compared. Salicylaldehyde was found to be the most cytotoxic followed by cinnamaldehyde and both rapidly depleted some glutathione before an inhibition of respiration occurred, which preceded cell lysis. Reactive oxygen species were formed, but lipid peroxidation was induced with cinnamaldehyde, but not salicylaldehyde. Glutathione depleted hepatocytes were more susceptible to cytotoxicity. Mitochondrial toxicity and cytotoxicity were prevented by glycolytic substrates (e.g. fructose), citric acid cycle substrates (e.g. glutamine) or cyclosporin, the mitochondrial permeability transition inhibitor. Inhibition of mitochondrial ALDH with chloral hydrate, crotonaldehyde or citral or decreasing mitochondrial NAD+ with rotenone increased cinnamaldehyde induced cytotoxicity with a much smaller effect on salicylaldehyde induced cytotoxicity. Cyanamide was the most effective ALDH inhibitor for increasing cinnamaldehyde induced cytotoxicity, presumably because cyanamide also inhibits microsomal ALDH. Although cinnamaldehyde was a better substrate than salicylaldehyde for ADH1, cytosolic NADH generators (e.g. xylitol) prevented salicylaldehyde and cinnamaldehyde cytotoxicity similarly. This could be explained as salicylaldehyde was not a substrate for the ALDHs and would then be more dependent on ADH for detoxification.

Glutathione Carbamoylation with S-Methyl N, N-diethylthiolcarbamate Sulfoxide and Sulfone : Mitochondrial Low Km Aldehyde Dehydrogenase Inhibition and Implications for Its Alcohol-Deterrent Action

S-Methyl N, N-diethylthiolcarbamate sulfoxide (DETC-MeSO) and sulfone (DETC-MeSO 2) both inhibit rat liver low K m aldehyde dehydrogenase (ALDH 2) in vitro and in vivo (Nagendra et al., Biochem Pharmacol 47: 1465–1467, 1994). DETC-MeSO has been shown to be a metabolite of disulfiram, but DETC-MeSO 2 has not. Studies were carried out to further investigate the inhibition of ALDH 2 by DETC-MeSO and DETC-MeSO 2. In an in vitro system containing hydrogen peroxide and horseradish peroxidase, the rate of DETC-MeSO oxidation corresponded to the rate of DETC-MeSO 2 formation. Carbamoylation of GSH by both DETC-MeSO and DETC-MeSO 2 was observed in a rat liver S 9 fraction. Carbamoylation of GSH was not observed in the presence of N-methylmaleimide. In in vitro studies, DETC-MeSO and DETC-MeSO 2 were equipotent ALDH 2 inhibitors when solubilized mitochondria were used, but DETC-MeSO was approximately four times more potent than DETC-MeSO 2 in intact mitochondria. In studies with rats, the dose (i.p. or oral) required to inhibit 50% ALDH 2 ( ed 50) was 3.5 mg/kg for DETC-MeSO and approximately 35 mg/kg for DETC-MeSO 2, approximately a 10-fold difference. Furthermore, maximum ALDH 2 inhibition occurred 1 hr after DETC-MeSO administration, whereas maximal ALDH 2 inhibition occurred 8 hr after DETC-MeSO 2 dosing. DETC-MeSO is, therefore, not only a more potent ALDH 2 inhibitor than DETC-MeSO 2 in vivo, but also in vitro when intact mitochondria are utilized. The in vitro results thus support the in vivo findings. Since oxidation of DETC-MeSO can occur both enzymatically and non-enzymatically, it is possible that DETC-MeSO 2 is formed in vivo. DETC-MeSO 2, however, is not as effective as DETC-MeSO in inhibiting ALDH 2, probably because it has difficulty penetrating the mitochondrial membrane. Thus, even if DETC-MeSO 2 is formed in vivo from DETC-MeSO, it is the metabolite DETC-MeSO that is most likely responsible for the inhibition of ALDH 2 after disulfiram administration.

Inhibition of recombinant human mitochondrial and cytosolic aldehyde dehydrogenases by two candidates for the active metabolites of disulfiram.

We expressed recombinant human cytosolic (ALDH1, high Km) and mitochondrial aldehyde dehydrogenase (ALDH2, low Km) in Escherichia coli and purified the enzymes to homogeneity to examine the nature of inhibition of human ALDH by disulfiram, its confirmed metabolite S-methyl N,N-diethylthiocarbamate (MeDTC) sulfoxide, and its proposed metabolite MeDTC sulfone. Disulfiram, MeDTC sulfoxide, and MeDTC sulfone, respectively, were potent inhibitors with IC50 values of 0.15 +/- 0.02 microM, 0.27 +/- 0.04 microM, and 0.12 +/- 0.02 microM for ALDH1, and 1.45 +/- 0.40 microM, 1.16 +/- 0.56, and 0.40 +/- 0.10 microM for ALDH2. Extensive dialysis did not restore the activity of the inactivated enzyme, indicating irreversible inhibition. Both the esterase and dehydrogenase activities of ALDH2 were inhibited to the same extent by MeDTC sulfone and sulfoxide, suggesting that both catalytic sites are closely linked. The time course of inhibition of ALDH appeared to be first-order for both MeDTC sulfone and MeDTC sulfoxide. Kitz and Wilson plots of the half-life of inactivation versus 1/[inhibitor] indicated that the reactions between ALDH and inhibitors were bimolecular. The pseudobimolecular rate constants (k3/KI) for the ALDH-inhibitor reactions were 1 x 10(5), 1 x 10(4), 3 x 10(3), and 1 x 10(3) s-1 M-1 ALDH1-sulfone, ALDH1-sulfoxide, ALDH2-sulfone, and ALDH2-sulfoxide, respectively. ALDH2 was not significantly protected from inactivation from either MeDTC sulfoxide or MeDTC sulfone by NAD alone, but high concentrations of NAD and acetaldehyde completely prevented inhibition. Since disulfiram is rapidly metabolized in vivo, it is believed that disulfiram is too short-lived to inhibit ALDH directly. The results of our study indicate that MeDTC sulfoxide and sulfone are potent inhibitors of human ALDH and are reasonable candidates for the proximal inhibitors of ALDH following disulfiram administration.

S-Methyl N, N-diethylthiolcarbamate sulfone, an in vitro and in vivo inhibitor of rat liver mitochondrial low Km aldehyde dehydrogenase

S-Methyl N, N-diethylthiolcarbamate sulfone (DETC-Me sulfone) was investigated for its rat liver mitochondrial low K m aldehyde dehydrogenase (ALDH 2) inhibitory properties. DETC-Me sulfone inhibited ALDH 2 in vitro ( ic 50 = 3.8 μ M ) and in vivo ( id 50 = 170 μ mol/kg; 31 mg/kg ). Maximum inhibition (60%) of ALDH 2 was observed 8 hr after DETC-Me sulfone administration. In addition, incubation of S-methyl N, N-diethylthiolcarbamate (DETC-Me) or S-methyl N, N-diethylthiolcarbamate sulfoxide (DETC-Me sulfoxide) with rat liver microsomes and an NADPH-generating system failed to produce DETC-Me sulfone. Furthermore, DETC-Me sulfone could not be detected in plasma from rats treated with either DETC-Me sulfoxide or DETC-Me sulfone. In conclusion, DETC-Me sulfone inhibited ALDH 2 in vitro and in vivo. However, there was no evidence suggesting that DETC-Me sulfoxide was metabolized to DETC-Me sulfone.

Inhibition of aldehyde dehydrogenases by methylene blue

The effect of the redox dye methylene blue on the stability of NADH and on the activity of the enzyme aldehyde dehydrogenase (ALDH; EC 1.2.1.3) was examined. NADH was measured by HPLC with fluorometric or spectrophotometric detection. The ALDH activity assays were carried out by following the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) from 3,4-dihydroxyphenylacetaldehyde (DOPAL) using HPLC and electrochemical detection. Incubation of NADH solutions in the presence of methylene blue resulted in a time-dependent direct oxidation of NADH. Methylene blue inhibited the human erythrocyte and leukocyte ALDHs and the rat liver mitochondrial low- K m ALDH in a concentration-dependent manner. The inactivation was reversible by dilution, and kinetic analysis indicated that methylene blue inhibits the rat liver mitochondrial low- K m and human erythrocyte ALDHs competitively with respect to DOPAL, while no effect of the NAD + concentration was apparent. For the rat liver low- K m ALDH, a K i of 8.4 ± 2.8 μM (mean ± SD; N = 5) was calculated. The inhibition of ALDH and the resulting decrease in the redox effect on the NAD system bound to alcohol dehydrogenase (EC 1.1.1.1) could explain the protective effect of methylene blue against metabolic redox effects of ethanol.

Nitroxyl analogs as inhibitors of aldehyde dehydrogenase: C-nitroso compounds

We previously postulated that the catalase-mediated oxidation of cyanamide leads to the formation of the unstable intermediate, N-hydroxycyanamide, which spontaneously decomposes to nitroxyl, the putative inhibitor of aldehyde dehydrogenase (EC 1.2.1.3; AlDH). Since it was not possible to provide direct evidence for the inhibition of AlDH by nitroxyl, we examined the activity of three representative substituted nitroxyls (C-nitroso compounds), viz. nitrosobenzene (NB), 1-nitroso-adamantane (NA), and 2-methyl-2-nitrosopropane (MNP), as direct inhibitors of yeast A1DH in vitro. While NB and NA were highly effective inhibitors in this system exhibiting IC 50 values of 2.5 and 8.6 μM, respectively, MNP was considerably less effective with an IC 50 of 0.15 mM. When tested in vivo, NA did not show any inhibitory activity on the hepatic AlDH, possibly due to the lack of site-specific delivery of the active monomeric form of this compound. However, NB at a low dose did inhibit hepatic AlDH as reflected by an increase in blood acetaldehyde levels. These results attest to the abilities of NB and NA to act as direct inhibitors of A1DH analogous to nitroxyl itself.

Diazepam- and chlordiazepoxide-mediated increases in erythrocyte aldehyde dehydrogenase activity and its possible implications

Erythrocyte ALDH activity was assayed in alcoholic ( n = 70) and nonalcoholic ( n = 40) subjects. In general, alcoholics without any prior medications ( n = 57) were found to have a decreased ALDH activity (mean ± SD: 3.38 ± 1.7 mU; p < 0.001) as compared to control group (5.10 ± 1.57 mU). However, a group of alcoholics who were detoxified with benzodiazepines ( n = 13) prior to blood collection for enzyme assay were found to have higher ALDH activity (4.92 ± 2.46 mU; p < 0.05) as compared to alcoholics who were not detoxified. In vitro experiments demonstrated that both diazepam (DZM) and chlordiazepoxide (CDP) could activate the ALDH. The magnitude of enzyme activation by DZM and CDP appear to correlate with their relative potency of tranquilizing effect. Further, the observed ability of DZM to reverse the inhibition of ALDH mediated by disulfiram may explain the biochemical basis of the reported ability of benzodiazepines (BDZ) to reduce the intensity of disulfiram ethanol reaction (DER).

In vitro and in vivo inhibition of rat liver aldehyde dehydrogenase by s-methyl N, N-diethylthiolcarbamate sulfoxide, a new metabolite of disulfiram

In summary, these data provide the first evidence that DETC-MeSO is a natural metabolite of disulfiram, and a potent inhibitor of rat liver mitochondrial low Km ALDH both in vitro and in vivo. It is therefore proposed that, based upon evidence to date, DETC-MeSO appears to be the chemical species to which disulfiram must be bioactivated, and is the metabolite most likely responsible for disulfiram's inhibition of rat liver mitochondrial low Km ALDH in vivo. Characterization of the properties of DETC-MeSO as the metabolite responsible for disulfiram's action as an ALDH inhibitor is presently in the process of being completed.

Use of Leukocyte Aldehyde Dehydrogenase Activity to Monitor Inhibitory Effect of Disulfiram Treatment

Aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity was determined in leukocytes and erythrocytes from alcoholic patients during different stages of disulfiram (Antabuse) treatment. Assays were performed by incubating intact isolated blood cells in phosphate-buffered saline (pH 7.4, 37 degrees C), using 3,4-dihydroxyphenylacetaldehyde (DOPAL), the biogenic aldehyde derived from dopamine, as substrate. The ALDH activity was assessed from the amount of 3,4-dihydroxyphenylacetic acid (DOPAC) formed, as analysed by high-performance liquid chromatography with electrochemical detection. The leukocyte ALDH, which is similar to the liver "mitochondrial" low-Km ALDH isozyme, was maximally inhibited (about 40-60%) within 2 to 3 days after the initial disulfiram administration (dosage 200 or 400 mg/day orally). The time to reach maximum inhibition (about 95%) of the erythrocyte ALDH, which closely resembles the liver "cytosolic" high-Km isozyme, varied from 3 to more than 6 days. When medication was completed, the leukocyte ALDH activity remained unaltered for the first 2 days, and did not revert to normal levels until about 6 to 7 days after terminating treatment. The erythrocyte ALDH was still inhibited by about 90% 1 week after the last disulfiram administration. These results suggest that the leukocyte ALDH activity might provide an easily accessible marker for monitoring effect and time course of ALDH inhibition during disulfiram treatment.

Inhibition of human erythrocyte and leukocyte aldehyde dehydrogenase activities by diethylthiocarbamic acid methyl ester: An in vivo metabolite of disulfiram

The inhibitory effects of diethylthiocarbamic acid methyl ester (DTC-Me), an in vivo metabolite ofdisulfiram (Antabuse®), on the aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activities in human erythrocytes and leukocytes were studied. ALDH assays were performed by incubating intact isolated blood cells in the presence of different concentrations of DTC-Me, using 3,4-dihydroxy-phenylacetaldehyde, the aldehyde derived from dopamine, as the substrate. DTC-Me was more selective as inhibitor of the leukocyte ALDH activity (which resembles the liver “mitochondrial” low ifK m ALDH), whereas both disulfiram and diethyldithiocarbamic acid, the reduced monomer of disulfiram, were more selective for the erythrocyte ALDH (which is similar to the “cytosolic” high- K m . ALDH). Diethylthiocarbamic acid, the free add of DTC-Me, was less potent than DTC-Me, and caused similar inactivation of the erythrocyte and leukocyte ALDH activities. The inhibition of ALDH by DTC-Me could not be completely restored by extensive dilution of intact or sonicated blood cell samples, which indicated that ALDH was irreversibly inhibited. Since the inhibition patterns with DTC-Me agrees with the previously reported patterns of inhibition of the high- K m and low- K m isozymes after the administration of disulfiram, the results suggest that DTC-Me might be the active in vivo inhibitory metabolite of disulfiram.

The mechanism of alcohol intolerance produced by various therapeutic agents.

According to clinical reports, several therapeutic agents produce ethanol intolerance, which is often referred as disulfiram reaction. The mechanism of this manifestation was investigated in the Wistar rat, by measuring the alcohol and aldehyde dehydrogenases (ADH, ALDH) of the liver and the brain after subacute administration of chloramphenicol (100 mg/kg X 4, intraperitoneally), chlorpropamide (80 mg/kg X 4, intraperitoneally), disulfiram (150 mg/kg X 4, intraperitoneally), griseofulvin (100 mg/kg X 4, intraperitoneally), isoniazid (200 mg/kg X 4, intraperitoneally), metronidazole (200 mg/kg X 4, intraperitoneally), and procarbazine (100 mg/kg X 4, intraperitoneally). All substances tested decrease the activity of the low-Km ALDH in the brain, with the exception of griseofulvin. The hepatic low-Km enzyme is also inhibited, with the exception of griseofulvin and metronidazole. The high-Km ALDH responds in an inconsistent way, while ADH is not affected at all. The results suggest that the so-called "disulfiram-reaction" is mediated mainly, but not exclusively, by inhibition of the low-Km ALDH.