Inhibition of aldehyde dehydrogenase by disulfiram and its metabolite methyl diethylthiocarbamoyl-sulfoxide.

Disulfiram (DSF) is presently the only available drug used in the aversion therapy of recovering alcoholics. It acts by inhibiting aldehyde dehydrogenase (ALDH), leading to high blood levels of acetaldehyde. The in vitro inhibition of ALDH by DSF and its metabolites was systematically studied by combined enzyme inhibition assay with direct molecular weight determination of the same sample using electrospray ionization-mass spectrometry (ESI-MS). Enzyme activity was measured after incubating yeast ALDH (yALDH) with excess concentrations of DSF, methyl diethyldithiocarbamate (MeDDC) and methyl diethylthiocarbamoyl-sulfoxide (MeDTC-SO) and then subjected to analysis by ESI-MS. Addition of DSF resulted in complete enzyme inhibition; however, ESI-MS analysis demonstrated no discernible shift in molecular weight, indicating that no intermolecular adduct was formed with the protein. Treatment of yALDH with MeDTC-SO also completely abolished yALDH activity with a concomitant increase of + approximately 100 Da in the molecular mass of the enzyme. This indicated formation of a covalent carbamoyl protein adduct. Furthermore, the effects of dithiothreitol (DTT) were examined on samples of inhibited protein in vitro. At pH 7.5, DTT completely reversed inhibition after DSF treatment. yALDH inhibited by MeDTC-SO could not be recovered by DTT at pH 7.5, but at pH 9 the enzymic activity was fully restored and a mass loss of approximately 100 Da was noted. This observations are consistent with mechanisms where inhibition of yALDH by DSF in vitro involves oxidation of the active site, whereas MeDTC-SO forms a covalent adduct with the protein in vitro resulting in cessation of enzyme activity.

S-methyl N,N-diethylthiocarbamate sulfone, a potential metabolite of disulfiram and potent inhibitor of low Km mitochondrial aldehyde dehydrogenase.

Disulfiram inhibits hepatic aldehyde dehydrogenase (ALDH) causing an accumulation of acetaldehyde after ethanol ingestion. It is thought that disulfiram is too short-lived in vivo to directly inhibit ALDH, but instead is biotransformed to reactive metabolites that inhibit the enzyme. S-Methyl N,N-diethylthiocarbamate (MeDTC) sulfoxide has been identified in the blood of animals given disulfiram and is a potent inhibitor of ALDH (Hart and Faiman, Biochem Pharmacol 46: 2285-2290, 1993). MeDTC sulfone is a logical metabolite of MeDTC sulfoxide. Therefore, we investigated the effects of MeDTC sulfone on the activity of rat hepatic low Km mitochondrial ALDH, the major enzyme in the metabolism of acetaldehyde. MeDTC sulfone inhibited the low Km mitochondrial ALDH in vitro with an IC50 of 0.42 +/- 0.04 microM (mean +/- SD, N = 5) compared with disulfiram, which had an IC50 of 7.5 +/- 1.2 microM under the same conditions. The inhibition of ALDH by MeDTC sulfone was time dependent. The decline in ALDH activity followed pseudo first-order kinetics with an apparent half-life of 2.1 min at 0.6 microM MeDTC sulfone. Inhibition of ALDH by MeDTC sulfone was apparently irreversible; dilution of the inhibited enzyme did not restore lost activity. The substrate (acetaldehyde, 80 microM) and cofactor (NAD, 0.5 mM) together completely protected ALDH from inhibition by MeDTC sulfone; substrate alone partially protected the enzyme. Addition of either thiol-containing compound glutathione (GSH) or dithiothreitol (DTT) to MeDTC sulfone before incubation with the enzyme increased the IC50 of MeDTC sulfone by 7- to 14-fold. Neither GSH nor DTT could restore lost ALDH activity after exposure of the enzyme to MeDTC sulfone. Results of these studies indicate that MeDTC sulfone, a potential metabolite of disulfiram, is a potent, irreversible inhibitor of low Km mitochondrial ALDH.

The influence of recombinant human insulin-like growth factor-I (rhIGF-I) on cell growth and cytotoxicity of drugs in childhood rhabdomyosarcoma cell lines and xenograft models.

PURPOSE: Recombinant human insulin-like growth factor I (rhIGF-I) has been reported to ameliorate vincristine-induced neuropathy, the dose-limiting side effect of this antimitotic anticancer drug. However, rhIGF-I also might have adverse effects, as has been shown in vitro, where it stimulates growth of cancer cells and protects them from cytotoxicity of anticancer drugs. The influence of rhIGF-I on the cytotoxicity of vincristine has not yet been studied. Furthermore, studies performed have been done under serum-free conditions, which are far from physiological. METHODS: We studied the influence of rhIGF-I on the growth of two rhabdomyosarcoma cell lines (Rh30 and Rh1) and on the antitumor effects of vincristine, cisplatin, etoposide, doxorubicin, and topotecan under serum-free and serum-containing conditions. To extend the in vitro data, we grew Rh30 cells as xenografts in mice and determined the effects of vincristine. rhIGF-I or their combination on tumor growth. RESULTS: In vitro, both cell lines demonstrated a functional type I IGF receptor, as shown by the rapid activation of ribosomal p70 S6 kinase after stimulation with rhIGF-I. Under serum-free conditions, rhIGF-I stimulated growth of both cell lines. Exposure to cytotoxic drugs with and without rhIGF-I resulted in higher cell numbers in cultures exposed to rhIGF-I. However, relative to the appropriate control, fractional growth inhibition and or cell kill of the cytotoxic drugs was identical with and without rhIGF-I. Under serum-containing conditions, rhIGF-I had no effect on cell growth or drug cytotoxicity. In vivo we did not find a significant influence of rhIGF-I on HxRh30 cell growth, or on the antitumor activity of vincristine. CONCLUSIONS: These studies show that rhIGF-I has no adverse effects on human rhabdomyosarcoma growth or on the antitumor effect of cytotoxic drugs under serum-containing conditions in vitro or in tumor-bearing mice. Potentially, therefore, rhIGF-I may ameliorate vincristine-induced neuropathy without adversely influencing tumor growth or vincristine cytotoxicity in children.

Simultaneous structure-activity determination of disulfiram photolysis products by on-line continuous-flow liquid secondary ion mass spectrometry and enzyme inhibition assay.

Disulfiram (DSF) is used in the treatment of recovering alcoholics and exerts its effect by inhibiting the enzyme aldehyde dehydrogenase (ALDH). We analyzed a mixture of products derived photochemically from DSF with on-line microbore HPLC-continuous-flow liquid secondary ion mass spectrometry (HPLC-CF-LSI-MS). By utilizing the post-HPLC column split of solvent flow, a small proportion (ca. 5%) was sent directly into the mass spectrometer, and the remainder was collected. Simultaneous MS analysis and enzyme inhibition studies on ALDH were then possible. Furthermore, using HPLC-CF-LSI-MS-MS, we were able to structurally characterize an interesting sulfine compound that inhibited ALDH.