Alcohol Abuse Drug Disulfiram Is Effective against Cyst Stages of Entamoeba histolytica Parasite.

New anti-Entamoeba histolytica multistage drugs are needed because only one drug class, nitroimidazoles, is available for treating invasive disease, and it does not effectively eradicate the infective cyst stage. Zinc ditiocarb (ZnDTC), a main metabolite of the FDA-approved drug disulfiram, was recently shown to be highly effective against the invasive trophozoite stage. In this brief report, we show that ZnDTC is active against cysts, with similar potency to first-line cysticidal drug paromomycin.

Triphenyltin(IV) dithiocarbamate compound induces genotoxicity and cytotoxicity in K562 human erythroleukemia cells primarily via mitochondria-mediated apoptosis.

The novel di-and triphenyltin(IV) dithiocarbamate compounds represented as RnSnL2 (where R = C4H9, C6H5; n = 2,3; L = N,N-dithiocarbamate), Ph2Sn(N,N-diisopropyldithiocarbamate) (OC1), Ph3Sn(N,N-diisopropyldithiocarbamate) (OC2), Ph2Sn(N,N-diallyldithiocarbamate) (OC3), Ph3Sn(N,N-diallyldithiocarbamate) (OC4), and Ph2Sn(N,N-diethyldithiocarbamate) (OC5) were assessed for their cytotoxicity in K562 human erythroleukemia cells. All compounds inhibited the growth of cells at low micromolar concentrations (<10 µM), and the mechanism underlying their antiproliferative effects on K562 cells was apoptosis, as corroborated by the exposure of plasma membrane phosphatidylserine. OC2, which showed the most promising antiproliferative activity, was selected for further analyses. The results demonstrated that OC2 induced apoptosis in K562 cells via an intrinsic mitochondrial pathway triggered upon DNA damage, an early apoptotic signal. Subsequently, OC2 produced excessive intracellular reactive oxygen species. The role of oxidative stress was corroborated by the significant reduction in GSH levels and percentage of apoptosis in NAC-pretreated cells. OC2 could arrest the cell cycle progression in the S phase. These new findings elucidate the antiproliferative potential of OC2 in the K562 human erythroleukemia cells and warrant further investigation, specifically to determine the exact signaling pathway underlying its antileukemic efficacy.

Seesaw conformations of Npl4 in the human p97 complex and the inhibitory mechanism of a disulfiram derivative.

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

Mechanism of cystathionine-ß-synthase inhibition by disulfiram: The role of bis(N,N-diethyldithiocarbamate)-copper(II).

BACKGROUND: Hydrogen sulfide (H2S) is an endogenous mammalian gasotransmitter. Cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) are the principal enzymes responsible for its biogenesis. A recent yeast screen suggested that disulfiram (a well-known inhibitor of aldehyde dehydrogenase and a clinically used drug in the treatment of alcoholism) may inhibit CBS in a cell-based environment. However, prior studies have not observed any direct inhibition of CBS by disulfiram. We investigated the potential role of bioconversion of disulfiram to bis(N,N-diethyldithiocarbamate)-copper(II) complex (CuDDC) in the inhibitory effect of disulfiram on H2S production and assessed its effect in two human cell types with high CBS expression: HCT116 colon cancer cells and Down syndrome (DS) fibroblasts. METHODS: H2S production from recombinant human CBS, CSE and 3-MST was measured using the fluorescent H2S probe AzMC. Mouse liver homogenate (a rich source of CBS) was also employed to measure H2S biosynthesis. The interaction of copper with accessible protein cysteine residues was evaluated using the DTNB method. Cell proliferation and viability were measured using the BrdU and MTT methods. Cellular bioenergetics was evaluated by Extracellular Flux Analysis. RESULTS: While disulfiram did not exert any significant direct inhibitory effect on any of the H2S-producing enzymes, its metabolite, CuDDC was a potent inhibitor of CBS and CSE. The mode of its action is likely related to the complexed copper molecule. In cell-based systems, the effects of disulfiram were variable. In colon cancer cells, no significant effect of disulfiram was observed on H2S production or proliferation or viability. In contrast, in DS fibroblasts, disulfiram inhibited H2S production and improved proliferation and viability. Copper, on its own, failed to have any effects on either cell type, likely due to its low cell penetration. CuDDC inhibited H2S production in both cell types studied and exerted the functional effects that would be expected from a CBS inhibitor: inhibition of cell proliferation of cancer cells and a bell-shaped effect (stimulation of proliferation at low concentration and inhibition of these responses at higher concentration) in DS cells. Control experiments using a chemical H2S donor showed that, in addition to inhibiting CBS and CSE, part of the biological effects of CuDDC relates to a direct reaction with H2S, which occurs through its complexed copper. CONCLUSIONS: Disulfiram, via its metabolite CuDDC acts as an inhibitor of CBS and a scavenger of H2S, which, in turn, potently suppresses H2S levels in various cell types. Inhibition of H2S biosynthesis may explain some of the previously reported actions of disulfiram and CuDDC in vitro and in vivo. Disulfiram or CuDDC may be considered as potential agents for the experimental therapy of various pathophysiological conditions associated with H2S overproduction.

DpdtC-Induced EMT Inhibition in MGC-803 Cells Was Partly through Ferritinophagy-Mediated ROS/p53 Pathway.

Epithelial-mesenchymal transition (EMT) is a cellular process in which epithelial cells are partially transformed into stromal cells, which endows the polarized epithelium cells more invasive feature and contributes cancer metastasis and drug resistance. Ferritinophagy is an event of ferritin degradation in lysosomes, which contributes Fenton-mediated ROS production. In addition, some studies have shown that ROS participates in EMT process, but the effect of ROS stemmed from ferritin degradation on EMT has not been fully established. A novel iron chelator, DpdtC (2,2'-di-pyridylketone dithiocarbamate), which could induce ferritinophagy in HepG2 cell in our previous study, was used to investigate its effect on EMT in gastric cancer cells. The proliferation assay showed that DpdtC treatment resulted in growth inhibition and morphologic alteration in MGC-803 cell (IC50 = 3.1 ± 0.3 µM), and its action involved ROS production that was due to the occurrence of ferritinophagy. More interestingly, DpdtC could also inhibit EMT, leading to the upregulation of E-cadherin and the downregulation of vimentin; however, the addition of NAC and 3-MA could attenuate (or neutralize) the action of DpdtC on ferritinophagy induction and EMT inhibition, supporting that the enhanced ferritinophagic flux contributed to the EMT inhibition. Since the degradation of ferritin may trigger the production of ROS and induce the response of p53, we next studied the role of p53 in the above two-cell events. As expected, an upregulation of p53 was observed after DpdtC insulting; however, the addition of a p53 inhibitor, PFT-α, could significantly attenuate the action of DpdtC on ferritinophagy induction and EMT inhibition. In addition, autophagy inhibitors or NAC could counteract the effect of DpdtC and restore the level of p53 to the control group, indicating that the upregulation of p53 was caused by ferritinophagy-mediated ROS production. In conclusion, our data demonstrated that the inhibition of EMT induced by DpdtC was realized through ferritinophagy-mediated ROS/p53 pathway, which supported that the activation of ferritinophagic flux was the main driving force in EMT inhibition in gastric cancer cells, and further strengthening the concept that NCOA4 participates in EMT process.

Vitamin B12b Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells.

We have shown that hydroxycobalamin (vitamin В12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + В12b. It was found that cell death induced by DDC + B12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.

Hydroxycobalamin catalyzes the oxidation of diethyldithiocarbamate and increases its cytotoxicity independently of copper ions.

It is known that some metals (Cu, Zn, Cd, Au) markedly increase the toxic effect of thiocarbamates. It was shown in the present study that hydroxycobalamin (a form of vitamin B12, HOCbl), which incorporates cobalt, significantly enhances the cytotoxicity of diethyldithiocarbamate (DDC), decreasing its IC50 value in tumor cells three to five times. The addition of HOCbl to aqueous DDC solutions accelerated the reduction of oxygen. No hydrogen peroxide accumulation was observed in DDC + HOCbl solutions; however, catalase slowed down the oxygen reduction rate. Catalase as well as the antioxidants N-acetylcysteine (NAC) and glutathione (GSH) partially inhibited the cytotoxic effect of DDC + HOCbl, whereas ascorbate, pyruvate, and tiron, a scavenger of superoxide anion, had no cytoprotective effect. The administration of HOCbl into DDC solutions (> 1 mM) resulted in the formation of a crystalline precipitate, which was inhibited in the presence of GSH. The data of UV and NMR spectroscopy and HPLC and Mass Spectrometry (LC/MS) indicated that the main products of the reaction of DDC with HOCbl are disulfiram (DSF) and its oxidized forms, sulfones and sulfoxides. The increase in the cytotoxicity of DDC combined with HOCbl occurred both in the presence of Cu2+ in culture medium and in nominally Cu-free solutions, as well as in growth medium containing the copper chelator bathocuproine disulfonate (BCS). The results indicate that HOCbl accelerates the oxidation of DDC with the formation of DSF and its oxidized forms. Presumably, the main cause of the synergistic increase in the toxic effect of DDC + HOCbl is the formation of sulfones and sulfoxides of DSF.

The binuclear form of dinitrosyl iron complexes with thiol-containing ligands in animal tissues.

It has been established that treatment of mice with sodium nitrite, S-nitrosoglutathione and the water-soluble nitroglycerine derivative isosorbide dinitrate (ISDN) as NO donors initiates in vivo synthesis of significant amounts of EPR-silent binuclear dinitrosyl iron complexes (B-DNIC) with thiol-containing ligands in the liver and other tissues of experimental mice. This effect is especially apparent if NO donors are administered to mice simultaneously with the Fe2+-citrate complex. Similar results were obtained in experiments on isolated liver and other mouse tissues treated with gaseous NО in vitro and during stimulation of endogenous NO synthesis in the presence of inducible NO synthase. B-DNIC appeared in mouse tissues after in vitro treatment of tissue samples with an aqueous solution of diethyldithiocarbamate (DETC), which resulted in the transfer of iron-mononitrosyl fragments from B-DNIC to the thiocarbonyl group of DETC and the formation of EPR-detectable mononitrosyl iron complexes (MNIC) with DETC. EPR-Active MNIC with N-methyl-d-glucamine dithiocarbamate (MGD) were synthesized in a similar way. MNIC-MGD were also formed in the reaction of water-soluble MGD-Fe2+ complexes with sodium nitrite, S-nitrosoglutathione and ISDN.

Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development.

The development of copper-drug complexes (CDCs) is hindered due to their very poor aqueous solubility. Diethyldithiocarbamate (DDC) is the primary metabolite of disulfiram, an approved drug for alcoholism that is being repurposed for cancer. The anticancer activity of DDC is dependent on complexation with copper to form copper bis-diethyldithiocarbamate (Cu(DDC)2), a highly insoluble complex that has not been possible to develop for indications requiring parenteral administration. We have resolved this issue by synthesizing Cu(DDC)2 inside liposomes. DDC crosses the liposomal lipid bilayer, reacting with the entrapped copper; a reaction that can be observed through a colour change as the solution goes from a light blue to dark brown. This method is successfully applied to other CDCs including the anti-parasitic drug clioquinol, the natural product quercetin and the novel targeted agent CX-5461. Our method provides a simple, transformative solution enabling, for the first time, the development of CDCs as viable candidate anticancer drugs; drugs that would represent a brand new class of therapeutics for cancer patients.

Uptake of dissolved organic carbon-complexed 65Cu by the green mussel Perna viridis.

Stable Cu isotope ((65)Cu) was complexed with various representative dissolved organic carbon (DOC) types, including coastal seawater DOC, fulvic acid (FA), cyanobacteria spirulina (SP) DOC, histidine (His), cysteine (Cys), and lipophilic diethyl dithiocarbamate (DDC) at different concentrations. The uptake of these dissolved Cu species by the coastal green mussel Perna viridis was quantified for the first time. Copper complexed with different DOC types were taken up in some measure by mussels, depending on the DOC types. However, complexation generally reduced Cu uptake as compared to that of inorganic Cu species, and DOC type-specific negative relationships were found between DOC levels and Cu uptake. Strong Cu binding sites (including His and organic sulfur functional groups) within DOC appeared to control the inhibitory effects of DOC on Cu uptake, possibly due to the competitive binding of Cu between the dissolved phase and biological membranes. Therefore, differences in strong Cu binding site levels may explain the differences in bioavailability of Cu complexed with different types of DOC. At the same time, the variations in Cu-DOC uptake may also be partly attributed to the absorption of Cu-DOC complexes, especially for the small Cu-DOC complexes (e.g., Cu-Cys, Cu-His, or Cu-DDC). Our study highlights the importance of considering the specificity of Cu-DOC complexes when assessing biological exposure to dissolved Cu in natural waters, especially during events, such as phytoplankton bloom periods, that could modify DOC composition and concentrations.

In vitro metabolism, glutathione conjugation, and CYP isoform specificity of epoxidation of 4-vinylphenol.

4-Vinylphenol (4VP) has been identified as a minor urinary metabolite of styrene in rat and human volunteers. This compound has been shown to be more hepatotoxic and pneumotoxic than both styrene and styrene oxide at lower doses in rats and mice. To explore the possible toxicity mechanism of 4VP, the current study was conducted to investigate the metabolism of 4VP, the glutathione (GSH) conjugation of the metabolites of 4VP and its cytochrome P(450) (CYP) specificity in epoxidation in different microsomes in vitro. Incubations of 4VP with mouse lung microsomes afforded two major metabolites which were identified as 4-(2-oxiranyl)-phenol of 4VP (4VPO) and 4VP catechol. 4VPO was found to react with GSH to form GSH conjugate and 4VP catechol was found to further be metabolized to electrophilic species which react with GSH to form the corresponding 4VP catechol GSH conjugates. Relative formation rates for those GSH conjugates and the regioisomer formation of 4VPO-GSH conjugates with both inhibitors of CYP 2F2 and CYP 2E1 in microsomal incubation condition were also investigated. This present study provides better insight on the lung toxicity seen with 4VP, the toxic metabolite of commercial styrene.

Mechanism-based inactivation of human CYP2E1 by diethyldithocarbamate.

Although the ability of disulfiram to inactivate CYP2E1 has been known for more than 20 years, the mechanism has not yet been elucidated. A metabolite of disulfiram, diethyldithocarbamate (DDC), is converted by CYP2E1 to a reactive intermediate that subsequently inactivates the protein, leading to mechanism-based inactivation. Mass spectral analysis of the inactivated human 2E1 protein demonstrates that the inactivation is due to the formation of an adduct of the reactive metabolite of DDC with the apoprotein. These data, along with mass spectral analysis of a reactive intermediate trapped with GSH, indicate the involvement of a reactive intermediate with a molecular mass of 116 Da. Our results suggest that this binding involves formation of a disulfide bond with one of the eight cysteines in CYP2E1. The inactivation of wild-type CYP2E1 as well as two of its polymorphic mutants, CYP2E1*2 and CYP2E1*4, was also investigated. For wild-type CYP2E1, the K(I) was 12.2 µM and the k(inact) was 0.02 min(-1). The K(I) values for the two polymorphic mutants were 227.6 and 12.4 µM for CYP2E1.2 and CYP2E1.4, and the k(inact) values were 0.0061 and 0.0187 min(-1), respectively. These data indicate that DDC is a much less efficient inactivator of CYP2E1.2 than it is of either the wild-type or the CYP2E1.4 variant.

Contribution of aldehyde dehydrogenase 3A1 to disulfiram penetration through monolayers consisting of cultured human corneal epithelial cells.

We previously prepared 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) solutions containing disulfiram (DSF) and hydroxypropylmethylcellulose (HPMC, DSF solutions), and found the instillation of this DSF solutions delayed lens opacification in ICR/f rats, a recessive-type hereditary cataractous strain. In this study, we determined the corneal penetration mechanism of DSF solutions using human cornea epithelial cell monolayers based on the immortalized human cornea epithelial cell line (HCE-T) developed by Tropainen et al. [Invest. Ophthalmol. Vis. Sci., 42, 2942-2948 (2001)]. The transepithelial electrical resistance (TER) values of HCE-T cells increases from approximately 275 to 388 Omega.cm(2) by exposure to an air-liquid interface for 2 weeks. The penetration of DSF into the basolateral chamber was prevented by the increase in TER values. The DSF in solution was converted to diethyldithiocarbamate (DDC) during the penetration experiment using HCE-T cell monolayers, and a close relationship between the penetration coefficient of DDC and aldehyde dehydrogenase (ALDH) 3A1 mRNA expression (y=41.202x+18.587, R=0.9413) was observed. In addition, an anti-ALDH3A1 antibody significantly inhibited the DSF-DDC conversion. These results suggest that DSF in DSF solutions is converted to DDC via catalysis by an ALDH3A1 in the cornea, and then transited from the apical side to the basolateral side.

Purification and characterization of phenoloxidase from clam Ruditapes philippinarum.

Using L-dihydroxyphenylalanine (L-DOPA) as a specific substrate, phenoloxidase (PO) from clam (Ruditapes philippinarum) was purified by Q Sepharose Fast Flow ion-exchange chromatography and Sephacryl S-100 gel-filtration, and characterized biochemically and enzymatically in this study. The molecular mass of PO in SDS-PAGE is about 76.9 kDa, and the prophenoloxidase (proPO) molecule, isolated as a monomeric protein, is 84.1 kDa. The PO molecule had a high oxidative activity, and the proPO molecule had almost no oxidative activity. The PO activity was optimal at pH 7.0 and temperature of 40 degrees C. The Km value of the PO for L-DOPA was 2.2 mmol l(-1). The PO was extremely sensitive to benzoic acid and sodium sulfite, very sensitive to citric acid, thio urea, 1-phenyl-2-thiourea and cysteine, but not sensitive to ascorbic acid. Combined with its specific enzyme activity on tyrosine and L-DOPA, it can be concluded that the Ruditapes PO is probably a kind of tyrosinase-type phenoloxidase. The PO activity was strongly inhibited by ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DETC), Zn2+, Ca2+ and Cu2+, as well as by Mg2+. The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu2+ on DETC-inhibited PO activity, indicate that Ruditapes PO is most probably a copper-containing metalloenzyme.

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.

Copper uptake is required for pyrrolidine dithiocarbamate-mediated oxidation and protein level increase of p53 in cells.

The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues.

Nitric oxide and iron overload. Limitations of ESR detection by DETC.

The ability of the ESR technique based on diethyldithiocarbamate (DETC) administration was studied as a suitable method to assess NO generation in vivo. The technique was successfully employed to measure NO generation after LPS treatment. DETC2-Fe-NO adducts were detected in liver homogenates of iron overloaded animals. When iron was administered to the animals simultaneously with LPS, NO-dependent signal increased 122%, but the content of NO2- and NO3- in sera was significantly lower (44%) as compared to LPS-treated rats. Iron dextran administration was responsible for a three-fold increase in the DETC2-Fe-NO content in non-LPS treated rats, while NOS activity and sera NO2- and NO3- levels remained unaffected. The adduct generation rate by a chemical NO-source was recorded in the presence of either control or iron overloaded homogenates supplemented with DETC in vivo. The exposure of liver homogenates to NO was performed either by the addition of 1 mM SNAP as NO donor or infusing an aqueous NO solution. In the presence of iron overloaded samples the adduct generation rate was 3.8-4.4-fold higher than in the presence of control samples. This effect restricts the applicability of the method to experimental conditions where iron levels remain constant, therefore it is not suitable for NO generation studies in experimental models where animals were subjected to iron overload.

Overview--in vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites.

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC(50)=36.4 microM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC(50)=4.62 microM). We isolate and identify a carbamoylated peptide at Cys(302) with the sequence FNQGQC(301)C(302)C(303). Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys(302), present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.

Preparation and in vivo ocular absorption studies of disulfiram solid dispersion.

Disulfiram, a dimer of diethyldithiocarbamate (DDC) which is a strong radical scavenger, is known to prevent cataract development. However, disulfiram is hardly absorbed from the cornea and its bioavailability is extremely low. In this study, we attempted to prepare disulfiram solid dispersion for the improvement of ocular bioavailability. Solid dispersions of disulfiram were prepared by either an evaporation method or a spray-drying method, using polyvinylpyrrolidone (PVP) as a carrier. Preparations were analyzed by scanning electron microscopy, powder X-ray diffractometry and differential scanning calorimetry, and confirmed to be a solid dispersion. The particle size of the solid dispersion prepared by the spray-drying method was smaller than the preparation by the evaporation method (spray-drying: 3.3+/-0.04 microm, evaporation: 34.3+/-18.0 microm). An in vivo ocular absorption experiment was conducted by instilling solid dispersions to rabbit eye and measuring the DDC in the aqueous humor. After instillation of disulfiram and PVP physical mixture, DDC was not detected in the aqueous humor. On the other hand, DDC appeared in the aqueous humor after the instillation of a solid dispersion. Maximal concentration and the area under the aqueous humor concentration-time curve were greater in the solid dispersion prepared by the spray-drying method than the preparation by the evaporation method. Disulfiram solid dispersion, especially prepared by the spray-drying method, improved ocular bioavailability.

Induction of apoptosis by thiuramdisulfides, the reactive metabolites of dithiocarbamates, through coordinative modulation of NFkappaB, c-fos/c-jun, and p53 proteins.

Prolinedithiocarbamate (PDTC) and diethyldithiocarbamate (DDTC) are cancer chemopreventive agents and can be biotransformed to prolinethiuramdisulfide (PTDS) and tetraethylthiuramdisulfide (disulfiram; DTDS), respectively. We found that the reactive metabolites PTDS and DTDS induced apoptosis after G1/S arrest. Phosphorylation of cyclin E, inhibition of cyclin-dependent kinase 2 activity, and degradation of cyclin E were found in human hepatoma Hep G2 cells during apoptosis. Moreover, PTDS and DTDS decreased the level of bcl-2 but increased the level of p53. In contrast, PDTC, DDTC, and ammonium dithiocarbamate (ADTC) did not induce apoptosis; rather they led to the induction of p53 and p21 followed by G1/S arrest. PDTC, DDTC, and ADTC also arrested cells in G1 phase. We then examined the effects of PTDS and DTDS on the signal transduction mechanisms leading to apoptosis. Although the transcription factors NFkappaB and AP-1 cooperatively decreased their DNA-binding activities to kappaB and 12-O-tetradecanoylphorbol-13-acetate-responsive elements, respectively, and p53 increased DNA-binding activity in the early stage but decreased it in the latter stage after treatment with PTDS, when the human Hep G2 cells were undergoing apoptosis. In summary, our results indicated that (i) PTDS and DTDS induced apoptosis and G1/S arrest mediated by p53, whereas PDTC, DDTC, and ADTC induced p53-dependent p21 expression leading to G1/S arrest; (ii) PDTC, DDTC, and ADTC induced p21/KIP1/CIP1 expression in a p53-dependent pathway leading to G1/S arrest; and (iii) NFkappaB, AP-1, and bcl-2 were downregulated during PTDS- and DTDS-induced apoptosis. These results suggested that PTDS and DTDS induced p53-dependent apoptosis, whereas PDTC, DDTC, and ADTC induced G1/S arrest. Apoptosis is regulated by the modulation of intracellular effectors such as NFkappaB, AP-1, and bcl-2 and activation of p53 in early stages.