The interaction of disulfiram and H2S metabolism in inhibition of aldehyde dehydrogenase activity and liver cancer cell growth.

Disulfiram (DSF), a sulfur-containing compound, has been used to treat chronic alcoholism and cancer for decades by inactivating aldehyde dehydrogenase (ALDH). Hydrogen sulfide (H2S) is a new gasotransmitter and regulates various cellular functions by S-sulfhydrating cysteine in the target proteins. H2S exhibits similar properties to DSF in the sensitization of cancer cells. The interaction of DSF and H2S on ALDH activity and liver cancer cell survival are not clear. Here it was demonstrated that DSF facilitated H2S release from thiol-containing compounds, and DSF and H2S were both capable of regulating ALDH through inhibition of gene expression and enzymatic activity. The supplement of H2S sensitized human liver cancer cells (HepG2) to DSF-inhibited cell viability. The expression of cystathionine gamma-lyase (a major H2S-generating enzyme) was lower but ALDH was higher in mouse liver cancer stem cells (Dt81Hepa1-6) in comparison with their parental cells (Hepa1-6), and H2S was able to inhibit liver cancer stem cell adhesion. In conclusion, these data point to the potential of combining DSF and H2S for inhibition of cancer cell growth and tumor development by targeting ALDH.

Mulberry leaves extract ameliorates alcohol-induced liver damages through reduction of acetaldehyde toxicity and inhibition of apoptosis caused by oxidative stress signals.

Mulberry leaves (Morus alba L.), which are traditional Chinese herbs, exert several biological functions, such as antioxidant, anti-inflammation, antidiabetic, and antitumor. Alcohol intake increases inflammation and oxidative stress, and this increase causes liver injury and leads to liver steatosis, cirrhosis, and hepatocellular carcinoma, which are major health problems worldwide. Previous report indicated that mulberry leaf extract (MLE) exited hepatoprotection effects against chronic alcohol-induced liver damages. In this present study, we investigated the effects of MLE on acute alcohol and liver injury induced by its metabolized compound called acetaldehyde (ACE) by using in vivo and in vitro models. Administration of MLE reversed acute alcohol-induced liver damages, increased acetaldehyde (ACE) level, and decreased aldehyde dehydrogenase activity in a dose-dependent manner. Acute alcohol exposure-induced leukocyte infiltration and pro-inflammation factors, including cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), were blocked by MLE in proportion to MLE concentration. MLE prevented alcohol-induced liver apoptosis via enhanced caveolin-1 expression and attenuated EGFR/STAT3/iNOS pathway using immunohistochemical analysis. ACE induced proteins, such as iNOS, COX-2, TNF-α, and IL-6, and inhibited superoxide dismutase expression, whereas co-treated with MLE reversed these proteins expression. MLE also recovered alcohol-induced apoptosis in cultured Hep G2 cells. Overall, our findings indicated that MLE ameliorated acute alcohol-induced liver damages by reducing ACE toxicity and inhibiting apoptosis caused by oxidative stress signals. Our results implied that MLE might be a potential agent for treating alcohol liver disease.

Optimized Dosing Schedule Based on Circadian Dynamics of Mouse Breast Cancer Stem Cells Improves the Antitumor Effects of Aldehyde Dehydrogenase Inhibitor.

Although malignant phenotypes of triple-negative breast cancer (TNBC) are subject to circadian alterations, the role of cancer stem cells (CSC) in defining this circadian change remains unclear. CSC are often characterized by high aldehyde dehydrogenase (ALDH) activity, which is associated with the malignancy of cancer cells and is used for identification and isolation of CSC. Here, we show that the population of ALDH-positive cells in a mouse 4T1 breast tumor model exhibits pronounced circadian alterations. Alterations in the number of ALDH-positive cells were generated by time-dependent increases and decreases in the expression of Aldh3a1 Importantly, circadian clock genes were rhythmically expressed in ALDH-negative cells, but not in ALDH-positive cells. Circadian expression of Aldh3a1 in ALDH-positive cells was dependent on the time-dependent release of Wingless-type mmtv integration site family 10a (WNT10a) from ALDH-negative cells. Furthermore, antitumor and antimetastatic effects of ALDH inhibitor N,N-diethylaminobenzaldehyde were enhanced by administration at the time of day when ALDH activity was increased in 4T1 tumor cells. Our findings reveal a new role for the circadian clock within the tumor microenvironment in regulating the circadian dynamics of CSC. These results should enable the development of novel therapeutic strategies for treatment of TNBC with ALDH inhibitors.Significance: This seminal report reveals that circadian dynamics of CSC are regulated by the tumor microenvironment and provides a proof of principle of its implication for chronotherapy in TNBC. Cancer Res; 78(13); 3698-708. ©2018 AACR.

Regulation of bioenergetics through dual inhibition of aldehyde dehydrogenase and mitochondrial complex I suppresses glioblastoma tumorspheres.

Background: Targeted approaches for treating glioblastoma (GBM) attempted to date have consistently failed, highlighting the imperative for treatment strategies that operate on different mechanistic principles. Bioenergetics deprivation has emerged as an effective therapeutic approach for various tumors. We have previously found that cancer cells preferentially utilize cytosolic NADH supplied by aldehyde dehydrogenase (ALDH) for ATP production through oxidative phosphorylation (OxPhos). This study is aimed at examining therapeutic responses and underlying mechanisms of dual inhibition of ALDH and OxPhos against GBM. Methods: For inhibition of ALDH and OxPhos, the corresponding inhibitors, gossypol and phenformin were used. Biological functions, including ATP levels, stemness, invasiveness, and viability, were evaluated in GBM tumorspheres (TSs). Gene expression profiles were analyzed using microarray data. In vivo anticancer efficacy was examined in a mouse orthotopic xenograft model. Results: Combined treatment of GBM TSs with gossypol and phenformin significantly reduced ATP levels, stemness, invasiveness, and cell viability. Consistently, this therapy substantially decreased expression of genes associated with stemness, mesenchymal transition, and invasion in GBM TSs. Supplementation of ATP using malate abrogated these effects, whereas knockdown of ALDH1L1 mimicked them, suggesting that disruption of ALDH-mediated ATP production is a key mechanism of this therapeutic combination. In vivo efficacy confirmed remarkable therapeutic responses to combined treatment with gossypol and phenformin. Conclusion: Our findings suggest that dual inhibition of tumor bioenergetics is a novel and effective strategy for the treatment of GBM.

Cyanamide phytotoxicity in soybean (Glycine max) seedlings involves aldehyde dehydrogenase inhibition and oxidative stress.

The phytotoxic effect of the allelochemical cyanamide has been well-documented yet the underlying mechanism for this phenomenon has not been fully characterized. Cognizant of the putative inhibitory effect of cyanamide on aldehyde dehydrogenases (ALDHs), we hereby show that the capacity of mitochondrial preparations from cyanamide-treated soybean seedlings to oxidize acetaldehyde and succinic-semialdehyde was dose-dependently reduced to at most 55% and 70%, respectively. Cyanamide-treated plants exhibited oxidative stress (i.e. increased lipid peroxidation and H2O2 accumulation) that was exacerbated upon exposure to UV-A--symptoms reminiscent of ALDH and succinic-semialdehyde dehydrogenase (SSADH) knock-out Arabidopsis mutants. We suggest that the inhibition of mitochondrial ALDH and SSADH may be a contributory mechanism to the burst in oxidative stress mediated by cyanamide.

Enrichment of chemical libraries docked to protein conformational ensembles and application to aldehyde dehydrogenase 2.

Molecular recognition is a complex process that involves a large ensemble of structures of the receptor and ligand. Yet, most structure-based virtual screening is carried out on a single structure typically from X-ray crystallography. Explicit-solvent molecular dynamics (MD) simulations offer an opportunity to sample multiple conformational states of a protein. Here we evaluate our recently developed scoring method SVMSP in its ability to enrich chemical libraries docked to MD structures of seven proteins from the Directory of Useful Decoys (DUD). SVMSP is a target-specific rescoring method that combines machine learning with statistical potentials. We find that enrichment power as measured by the area under the ROC curve (ROC-AUC) is not affected by increasing the number of MD structures. Among individual MD snapshots, many exhibited enrichment that was significantly better than the crystal structure, but no correlation between enrichment and structural deviation from crystal structure was found. We followed an innovative approach by training SVMSP scoring models using MD structures (SVMSPMD). The resulting models were applied to two difficult cases (p38 and CDK2) for which enrichment was not better than random. We found remarkable increase in enrichment power, particularly for p38, where the ROC-AUC increased by 0.30 to 0.85. Finally, we explored approaches for a priori identification of MD snapshots with high enrichment power from an MD simulation in the absence of active compounds. We found that the use of randomly selected compounds docked to the target of interest using SVMSP led to notable enrichment for EGFR and Src MD snapshots. SVMSP rescoring of protein-compound MD structures was applied for the search of small-molecule inhibitors of the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2). Rank-ordering of a commercial library of 50 000 compounds docked to MD structures of ALDH2 led to five small-molecule inhibitors. Four compounds had IC50s below 5 µM. These compounds serve as leads for the design and synthesis of more potent and selective ALDH2 inhibitors.

Inhibition of aldehyde dehydrogenase-2 suppresses cocaine seeking by generating THP, a cocaine use-dependent inhibitor of dopamine synthesis.

There is no effective treatment for cocaine addiction despite extensive knowledge of the neurobiology of drug addiction. Here we show that a selective aldehyde dehydrogenase-2 (ALDH-2) inhibitor, ALDH2i, suppresses cocaine self-administration in rats and prevents cocaine- or cue-induced reinstatement in a rat model of cocaine relapse-like behavior. We also identify a molecular mechanism by which ALDH-2 inhibition reduces cocaine-seeking behavior: increases in tetrahydropapaveroline (THP) formation due to inhibition of ALDH-2 decrease cocaine-stimulated dopamine production and release in vitro and in vivo. Cocaine increases extracellular dopamine concentration, which activates dopamine D2 autoreceptors to stimulate cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) in primary ventral tegmental area (VTA) neurons. PKA and PKC phosphorylate and activate tyrosine hydroxylase, further increasing dopamine synthesis in a positive-feedback loop. Monoamine oxidase converts dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a substrate for ALDH-2. Inhibition of ALDH-2 enables DOPAL to condense with dopamine to form THP in VTA neurons. THP selectively inhibits phosphorylated (activated) tyrosine hydroxylase to reduce dopamine production via negative-feedback signaling. Reducing cocaine- and craving-associated increases in dopamine release seems to account for the effectiveness of ALDH2i in suppressing cocaine-seeking behavior. Selective inhibition of ALDH-2 may have therapeutic potential for treating human cocaine addiction and preventing relapse.

Suppression of heavy drinking and alcohol seeking by a selective ALDH-2 inhibitor.

BACKGROUND: Inherited human aldehyde dehydrogenase 2 (ALDH-2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH-2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH-2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH-2 inhibitor might affect other metabolic factors involved in regulating drinking. METHODS: Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co-crystal structure of ALDH-2 and daidzin. We tested the efficacy of a highly selective reversible ALDH-2 inhibitor, CVT-10216, in models of moderate and high alcohol drinking rats. We studied 2-bottle choice and deprivation-induced drinking paradigms in Fawn Hooded (FH) rats, operant self-administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue-induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm. RESULTS: CVT-10216 increases acetaldehyde after alcohol gavage and inhibits 2-bottle choice alcohol intake in heavy drinking rodents, including deprivation-induced drinking. Moreover, CVT-10216 also prevents operant self-administration and eliminates cue-induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT-10216 prevents alcohol-induced increases in NAc DA without changing basal levels. CVT-10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses. CONCLUSION: Our findings suggest that selective reversible ALDH-2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.

Disulfiram metabolites permanently inactivate the human multidrug resistance P-glycoprotein.

The human multidrug resistance P-glycoprotein (P-gp) uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. The relatively high expression of P-gp in organs such as the intestine, kidney, blood-brain/testes barrier and in some tumor cells can compromise chemotherapy treatments for patients with cancer or AIDS/HIV. It has been difficult to inhibit P-gp during chemotherapy with noncovalent inhibitors because the relatively high levels of inhibitors have severe side effects. An alternative approach to inhibit P-gp would be to covalently modify cysteine residues within the NBDs. In this study, we tested whether metabolites of disulfiram, a drug currently used to treat chronic alcoholism, could inhibit P-gp. We show that the disulfiram metabolites, S-methyl N,N-diethylthiocarbamate sulfoxide and S-methyl N,N-diethylthiocarbamate sulfone inhibited the verapamil-stimulated ATPase activity of P-gp with IC50 values (concentrations that result in 50% inhibition of activity) of 9 and 4.8 microM, respectively. Similarly, S-methyl N,N-diethylthiocarbamate sulfoxide and S-methyl N,N-diethylthiocarbamate sulfone inhibited the activity of aldehyde dehydrogenase with IC50 values of 3.2 and 1.7 microM, respectively. Inhibition of P-gp by the metabolites was not reversed by addition of the reducing compound, dithiothreitol. We then determined which endogenous cysteine residue was responsible for inhibiting P-gp activity after exposure to the disulfiram metabolites. Treatment of P-gp mutants containing a single cysteine residue showed that inactivation was primarily due to modification of Cys1074 in NBD2. These results indicate that metabolites of disulfiram can covalently inactivate P-gp. Covalent modification of drug transporters could be a useful approach for inhibiting their activities during chemotherapy.

Discovery of novel targets of quinoline drugs in the human purine binding proteome.

The quinolines have been used in the treatment of malaria, arthritis, and lupus for many years, yet the precise mechanism of their action remains unclear. In this study, we used a functional proteomics approach that exploited the structural similarities between the quinoline compounds and the purine ring of ATP to identify quinoline-binding proteins. Several quinoline drugs were screened by displacement affinity chromatography against the purine binding proteome captured with gamma-phosphate-linked ATP-Sepharose. Screening of the human red blood cell purine binding proteome identified two human proteins, aldehyde dehydrogenase 1 (ALDH1) and quinone reductase 2 (QR2). In contrast, no proteins were detected upon screening of the Plasmodium falciparum purine binding proteome with the quinolines. In a complementary approach, we passed cell lysates from mice, red blood cells, or P. falciparum over hydroxychloroquine- or primaquine-Sepharose. Consistent with the displacement affinity chromatography screen, ALDH and QR2 were the only proteins recovered from mice and human red blood cell lysate and no proteins were recovered from P. falciparum. Furthermore, the activity of QR2 was potently inhibited by several of the quinolines in vitro. Our results show that ALDH1 and QR2 are selective targets of the quinolines and may provide new insights into the mechanism of action of these drugs.

Subchronic toxicity of chloral hydrate on rats: a drinking water study.

The subchronic toxicity of chloral hydrate, a disinfection byproduct, was studied in rats following 13 weeks of drinking water exposure. Male (262 +/- 10 g) and female (190 +/- 8 g) Sprague-Dawley rats, ten animals per group, were administered chloral hydrate via drinking water at 0.2, 2, 20 and 200 ppm. Control animals received distilled water only. Gross and microscopic examinations, serum chemistry, hematology, biochemical analysis, neurogenic amine analysis and serum trichloroacetic acid (TCA) analysis were performed at the end of the treatment period. Bronchoalveolar fluids were collected at necropsy and urine specimens were collected at weeks 2, 6 and 12 for biochemical analysis. No treatment-related changes in food and water intakes or body weight gains were observed. There were no significant changes in the weights of major organs. Except for a mild degree of vacuolation within the myelin sheath of the optic nerves in the highest dose males, there were no notable histological changes in the tissues examined. Statistically significant treatment-related effects were biochemical in nature, with the most pronounced being increased liver catalase activity in male rats starting at 2 ppm. Liver aldehyde dehydrogenase (ALDH) was significantly depressed, whereas liver aniline hydroxylase activity was significantly elevated in both males and females receiving the highest dose. A dose-related increase in serum TCA was detected in both males and females starting at 2 ppm. An in vitro study of liver ALDH confirmed that chloral hydrate was a potent inhibitor, with an IC(50) of 8 micro M, whereas TCA was weakly inhibitory and trichloroethanol was without effect. Analysis of brain biogenic amines was conducted on a limited number (n = 5) of male rats in the control and high dose groups, and no significant treatment-related changes were detected. Taking into account the effect on the myelin sheath of male rats and the effects on liver ALDH and aniline hydroxylase of both males and females at the highest dose level, the no-observed-effect level (NOEL) was determined to be 20 ppm or 1.89 mg kg(-1) day(-1) in males and 2.53 mg kg(-1) day(-1) in females. This NOEL is ca. 1000-fold higher than the highest concentration of chloral hydrate reported in the municipal water supply.

Rapid in vivo assay for topical oral cancer chemopreventive agents.

The cancer chemopreventive effect of topically applied phenethyl isothiocyanate (PEIT) was examined in a hamster buccal pouch model, in which squamous cell carcinomas (SCC) are induced at high frequency, by topical application of N-methyl-N-benzylnitrosamine (MBN). The buccal pouches of eleven hamsters were pretreated thrice-weekly for two weeks with corn oil (CO) containing 50 mM PEIT, followed by 22 weeks of twice-weekly application of CO containing MBN and PEIT, both at 50 mM. Under similar conditions, twelve hamsters were pretreated with CO for 2 weeks, followed by MBN in CO. Tumor analysis was performed 19 days after the last application of PEIT and MBN. The incidence of tumors (approximately 90% SCC) in the unprotected and protected groups was 100% and 73%, respectively. Although total tumor incidence was marginally decreased, PEIT inhibited significantly the tumor frequency and tumor burden by 79% and 74%, respectively. In both groups, 43% of the carcinomas exhibited p53 immunohistochemical activity. A short-term experiment was performed to determine whether PEIT inhibits MBN-induced cellular foci expressing gamma-glutamyltranspeptidase histochemical activity (gamma-GT foci). Buccal pouches of four protected hamsters received 50 mM PEIT pretreatment on days 1 and 4, followed on days 6 and 9 by application of CO containing MBN and PEIT, both at 50 mM. Four unprotected hamsters were similarly pretreated with CO, followed by MBN in CO. gamma-GT foci were enumerated in buccal pouch epithelial whole mounts prepared from pairs of protected and unprotected hamsters on days 10 through 13. Whereas the number of gamma-GT foci in unprotected hamsters ranged from 98 to 356 per 10 cm2, the protected hamsters exhibited no more than one focus per 10 cm2. This model may be useful for rapid identification of chemopreventive agents, and combinations of agents, which inhibit initiation and promotion stages of oral carcinogenesis.

Barbarea verna as a source of 2-phenylethyl glucosinolate, precursor of cancer chemopreventive phenylethyl isothiocyanate.

The isolation of gram-amounts of 2-phenylethyl glucosinolate (gluconasturtiin, GST) from Barbarea verna seeds is reported for the first time. This vegetable source was of crucial importance to isolate GST with a high purity grade and in high yield. Indeed, B. verna seeds contain GST as the only glucosinolate, unlike other sources. The availability at low cost of GST will allow further studies to explain the claimed anticancer activity of its derived phenylethyl isothiocyanate.

Cyanamide-induced activation of the hypothalamo-pituitary-adrenal axis.

The present study investigated the effects of acute administration of cyanamide (a potent inhibitor of aldehyde dehydrogenase used to treat alcoholics), on the hypothalamo-pituitary adrenal (HPA)-axis. Cyanamide resulted in a significant increase in arginine vasopressin mRNA and corticotrophin releasing factor (CRF) mRNA in the parvocellular cells of the paraventricular nucleus and pro-opiomelanocortin (POMC) mRNA in the anterior pituitary. Plasma corticosterone concentrations were elevated by a range of doses of cyanamide which were maintained in the high dose group at 4 h following administration. These results suggest that cyanamide is able to activate the HPA axis at all levels of the axis. Arginine vasopressin mRNA, in the parvocellular cells of the paraventricular nucleus is an important component of the stress response. Silver grain counting of emulsion dipped slides is commonly used for its evaluation following in-situ hybridization. This method is however, not entirely satisfactory and very time-consuming. We compared this method with a film autoradiographic method, and show that the film autoradiographic method is valid for the determination of arginine vasopressin mRNA in the parvocellular cells of the paraventricular nucleus.

Absolute bioavailability and absorption profile of cyanamide in man.

A pharmacokinetic study of cyanamide, an inhibitor of aldehyde dehydrogenase (EC1.2.1.3) used as an adjuvant in the aversive therapy of chronic alcoholism, has been carried out in healthy male volunteers following intravenous and oral administration. Cyanamide plasma levels were determined by a sensitive HPLC assay, specific for cyanamide. After intravenous administration cyanamide displayed a disposition profile according to a two-compartmental open model. Elimination half-life and total plasma clearance values ranged from 42.2 to 61.3 min and from 0.0123 to 0.0190 L.kg-1.min-1, respectively. After oral administration of 0.3, 1.0, and 1.5 mg/kg x +/- SEM values of Cmax, tmax (median) and AUC were 0.18 +/- 0.03, 0.91 +/- 0.11, and 1.65 +/- 0.27 micrograms.ml-1; 13.5, 13.5, and 12 min; and 8.59 +/- 1.32, 45.39 +/- 1.62, and 77.86 +/- 17.49 micrograms.ml-1.min, respectively. Absorption was not complete and the oral bioavailability, 45.55 +/- 9.22, 70.12 +/- 4.73, and 80.78 +/- 8.19% for the 0.3, 1.0, and 1.5 mg/kg doses, respectively, increased with the dose administered. The models that consider a first-order absorption process alone (whether with a fixed or variable bioavailability value as a function of dose) or with loss of drug due to presystemic metabolism (with zero-order or Michaelis-Menten kinetics) were simultaneously fitted to plasma level data obtained following 1 mg/kg i.v. and 0.3, 1.0, and 1.5 mg/kg oral administrations. The model that best fit the data was that with a first-order absorption process plus a loss by presystemic metabolism with Michaelis-Menten kinetics, suggesting the presence of a saturable first-pass effect.

Aldehyde dehydrogenase in tobacco pollen.

Acetaldehyde is one of the intermediate products of ethanolic fermentation, which can be reduced to ethanol by alcohol dehydrogenase (ADH). Alternatively, acetaldehyde can be oxidized to acetate by aldehyde dehydrogenase (ALDH) and subsequently converted to acetyl-CoA by acetyl-CoA synthetase (ACS). To study the expression of ALDHs in plants we isolated and characterized a cDNA coding for a putative mitochondrial ALDH (TobAldh2A) in Nicotiana tabacum. TobALDH2A shows 54-60% identity at the amino acid level with other ALDHs and shows 76% identity with maize Rf2, a gene involved in restoration of male fertility in cms-T maize. TobAldh2A transcripts and protein were present at high levels in the male and female reproductive tissues. Expression in vegetative tissues was much lower and no induction by anaerobic incubation was observed. This suggests that TobALDH expression is not part of the anaerobic response, but may have another function. The use of specific inhibitors of ALDH and the pyruvate dehydrogenase (PDH) complex indicates that ALDH activity is important for pollen tube growth, and thus may have a function in biosynthesis or energy production.

Glutathione- and glutathione-S-transferase-dependent oxidative desulfuration of the thione xenobiotic diethyldithiocarbamate methyl ester.

Oxidative desulfuration of diethyldithiocarbamate methyl ester (DDTC-Me), a thione xenobiotic and a metabolite of disulfiram, was studied. Using a rat liver microsomal incubation system, DDTC-Me was oxidized at the thionosulfur group, forming DDTC-Me sulfine. Only minimal desulfuration of DDTC-Me to S-methyl-N,N-diethylthiolcarbamate (DETC-Me) occurred. Desulfuration of DDTC-Me increased 4-fold when the microsomal incubation was supplemented with reduced glutathione (GSH) and increased 8-fold when both GSH and glutathione-S-transferase (EC 2.5.1.18) were added. Similar results were obtained using a simplified system containing DDTC-Me sulfine, GSH, and glutathione-S-transferase. This suggested that DDTC-Me sulfine is a stable intermediate formed before DDTC-Me is desulfurated to DETC-Me. This unprecedented desulfuration process can be explained as follows. GSH attacks the oxithiirane isomer of DDTC-Me sulfine, resulting in ring opening followed by loss of glutathione hydrodisulfide, which is reduced by GSH to oxidized glutathione and H2S. GSH can also reduce DDTC-Me sulfine to DDTC-Me. This mechanism is supported by in vitro studies. An approximately 1:1 stoichiometry was observed for the formation of H2S and DETC-Me. A 1:1 stoichiometry was also observed for the consumption of DDTC-Me sulfine, formation of DETC-Me plus DDTC-Me, and formation of oxidized glutathione. Glutathione hydrodisulfide was trapped by derivatization in situ using 4-vinylpyridine. Oxidative desulfuration of a series of dithiocarbamate esters also followed a similar mechanism.

Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase.

Human mitochondrial aldehyde dehydrogenase (ALDH-I) is potently, reversibly, and selectively inhibited by an isoflavone isolated from Radix puerariae and identified as daidzin, the 7-glucoside of 4',7-dihydroxyisoflavone. Kinetic analysis with formaldehyde as substrate reveals that daidzin inhibits ALDH-I competitively with respect to formaldehyde with a Ki of 40 nM, and uncompetitively with respect to the coenzyme NAD+. The human cytosolic aldehyde dehydrogenase isozyme (ALDH-II) is nearly 3 orders of magnitude less sensitive to daidzin inhibition. Daidzin does not inhibit human class I, II, or III alcohol dehydrogenases, nor does it have any significant effect on biological systems that are known to be affected by other isoflavones. Among more than 40 structurally related compounds surveyed, 12 inhibit ALDH-I, but only prunetin and 5-hydroxydaidzin (genistin) combine high selectivity and potency, although they are 7- to 15-fold less potent than daidzin. Structure-function relationships have established a basis for the design and synthesis of additional ALDH inhibitors that could both be yet more potent and specific.

Formation of the 37KD liver protein-acetaldehyde adduct in vivo and in vitro.

A liver protein with molecular weight of 37,000 can form adducts with acetaldehyde in vivo when rats are fed alcohol chronically. This 37KD protein is not directly involved in the hepatic metabolism of ethanol but it requires alcohol dehydrogenase activity to form adducts with acetaldehyde. The 37KD protein-AA is located in cytosol of the liver. However, under certain circumstances e.g. when fed an alcohol-containing liquid diet supplemented with cyanamide (an aldehyde dehydrogenase inhibitor that raises blood acetaldehyde concentrations), this 37KD protein-acetaldehyde adduct (protein-AA) becomes incorporated into liver plasma membranes. The same 37KD protein-AA can also form in vitro with cultured rat hepatocytes treated with ethanol. The formation of the 37KD protein-AA in the cultured liver cells increased with time and was dependent on concentrations of ethanol in the culture medium. Thus, protein-AAs can form in vivo and in liver cell culture upon chronic alcohol exposure, and a 37KD protein in liver is highly susceptible to chemical modification by acetaldehyde.

Lack of correlation between pharmacokinetic and pharmacodynamic behaviour of cyanamide in man. A preliminary report.

A pharmacokinetic and dynamic study of cyanamide, an inhibitor of aldehyde dehydrogenase (ALDH) used as an adjuvant in the aversive therapy of chronic alcoholism, has been carried out in man after oral administrations. Cyanamide plasma levels were determined by a sensitive and specific high performance liquid chromatographic assay. Blood ALDH activity were estimated after oral administration of 0.3, 1 and 1.5 mg/kg of cyanamide. One i.v. administration of 1 mg/kg was performed in order to determine the absolute bioavailability and the main pharmacokinetic parameters. Elimination half life and total plasma clearance values were 51.7 8.8 min and 14.4 2.7 mL/kg/min respectively. After oral administrations of 0.3, 1 and 1.5 mg/kg a rapid absorption rate was estimated with a Tmax values range of 10.5 to 15.5 min. The extent of absorption was not complete, oral bioavailability being 53%, 70% and 81% respectively. The presence of a first pass-effect is suggested. The inhibitory activity of cyanamide on blood ALDH reached the maximum value 4 h after its administration and decreased progressively throughout six days period. The cyanamide plasma levels time course did not correlated with the pharmacodynamic time course responses.