Richard E. Staub

University of California, Berkeley, Berkeley, California, United States

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Publications (6)22.33 Total impact

  • R E Staub, G B Quistad, J E Casida
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    ABSTRACT: Liver mitochondrial low-Km aldehyde dehydrogenase (ALDH2, EC 1.2.1.3), the isoform responsible for the conversion of acetaldehyde to acetate, is inhibited by the sulfoxide bioactivation products of Et2NC(O)SMe (from the alcohol aversion drug disulfiram), Pr2NC(O)SEt (the herbicide S-ethyl N,N-dipropylthiocarbamate), and BuNHC(O)SMe (from the fungicide benomyl). This study tested the hypothesis that bioactivated BuNHC(O)SMe, the most potent of these thiocarbamates, is a selective carbamoylating agent for ALDH2 of mouse liver in vivo and in vitro. [14C]BuNHC(O)SMe administered i.p. to mice labeled one principal mitochondrial protein, which cochromatographed with ALDH activity by in-gel assay after isoelectric focusing. The labeled protein was isolated by isoelectric focusing (pI 6.1) and SDS-PAGE (54 kDa) and identified as ALDH2 by sequencing of peptides from a tryptic digest. In vivo at 1.5 mg/kg, enzyme inhibition was 80%, and ALDH2 was the only mitochondrial protein labeled extensively, illustrating the outstanding potency and specificity. ALDH2 also was labeled upon incubation of mouse liver mitochondria with [14C]BuNH-C(O)SMe in the presence of microsomes (P450) and NADPH. In contrast, under similar conditions, [14C]Pr2NC(O)SEt sulfoxide labeled primarily two other proteins at approximately 58 and approximately 61 kDa, establishing a very different selectivity for the two sulfoxides. These findings are of interest relative to selective inhibitors and carbamoylating agents for ALDH2 and to alcohol aversion upon exposure to herbicides and fungicides.
    Biochemical Pharmacology 12/1999; 58(9):1467-73. · 4.65 Impact Factor
  • Richard E Staub, Gary B Quistad, John E Casida
    [Show abstract] [Hide abstract]
    ABSTRACT: Liver mitochondrial low-Km aldehyde dehydrogenase (ALDH2, EC 1.2.1.3), the isoform responsible for the conversion of acetaldehyde to acetate, is inhibited by the sulfoxide bioactivation products of Et2NC(O)SMe (from the alcohol aversion drug disulfiram), Pr2NC(O)SEt (the herbicide S-ethyl N,N-dipropylthiocarbamate), and BuNHC(O)SMe (from the fungicide benomyl). This study tested the hypothesis that bioactivated BuNHC(O)SMe, the most potent of these thiocarbamates, is a selective carbamoylating agent for ALDH2 of mouse liver in vivo and in vitro. [14C]BuNHC(O)SMe administered i.p. to mice labeled one principal mitochondrial protein, which cochromatographed with ALDH activity by in-gel assay after isoelectric focusing. The labeled protein was isolated by isoelectric focusing (pI 6.1) and SDS–PAGE (54 kDa) and identified as ALDH2 by sequencing of peptides from a tryptic digest. In vivo at 1.5 mg/kg, enzyme inhibition was 80%, and ALDH2 was the only mitochondrial protein labeled extensively, illustrating the outstanding potency and specificity. ALDH2 also was labeled upon incubation of mouse liver mitochondria with [14C]BuNHC(O)SMe in the presence of microsomes (P450) and NADPH. In contrast, under similar conditions, [14C]Pr2NC(O)SEt sulfoxide labeled primarily two other proteins at ∼58 and ∼61 kDa, establishing a very different selectivity for the two sulfoxides. These findings are of interest relative to selective inhibitors and carbamoylating agents for ALDH2 and to alcohol aversion upon exposure to herbicides and fungicides.
    Biochemical Pharmacology 11/1999; 58(9):1467-1473. DOI:10.1016/S0006-2952(99)00239-7 · 4.65 Impact Factor
  • Staub R.E, Quistad G.B, Casida J.E
    Biochemical Pharmacology 10/1999; 58(9):1467-1473. · 4.65 Impact Factor
  • Richard E. Staub, Gary B. Quistad, John E. Casida
    [Show abstract] [Hide abstract]
    ABSTRACT: Benomyl (a non-thio fungicide) inhibits hepatic mitochondrial low-Km aldehyde dehydrogenase (mALDH or ALDH2) in ip-treated mice by 50% (IC50) at 7.0 mg/kg, which is surprisingly the same potency range as that for several dithiocarbamate fungicides (and the related alcohol abuse drug disulfiram) and thiocarbamate herbicides previously known for their alcohol-sensitizing action. The mechanism by which benomyl inhibits mALDH was therefore examined, first by comparing the metabolism of benomyl with the aforementioned mono- and dithiocarbamates and second by evaluating the inhibitory potency of the benomyl metabolites. Benomyl in ip-treated mice is converted, via butyl isocyanate, S-(N-butylcarbamoyl)glutathione, and S-(N-butylcarbamoyl)cysteine, to S-methyl N-butylthiocarbamate (MBT), identified as a transient metabolite in liver. MBT is >10-fold more potent than benomyl or butyl isocyanate as an in vivo mALDH inhibitor and is also more potent than the intermediary S-(N-butylcarbamoyl) conjugates. Benomyl and MBT inhibit mouse hepatic mALDH in vitro with IC50s of 0.77 and 8.7 microM, respectively. The potency of MBT is greatly enhanced by fortification of the mitochondria with NADPH alone or plus microsomes giving IC50s of 0.50 and 0.23 microM, respectively. This activation of MBT is almost completely blocked by the cytochrome P450 inhibitor N-benzylimidazole but not by several other cytochrome P450 inactivators. MBT (probably following bioactivation) inhibits mALDH in vivo with an IC50 of 0.3 mg/kg. Two candidate activation products were synthesized for potency determinations. N-Hydroxy MBT (prepared via the trimethylsilyl derivative) was not detected as an MBT metabolite; its low potency also rules against N-hydroxylation as the activation process. MBT sulfoxide, from oxidation of MBT with magnesium monoperoxyphthalate in water, is one of the most potent inhibitors known for mALDH and yeast ALDH in vitro (IC50 0.08-0.09 microM). These findings are consistent with a six-step bioactivation of benomyl, via the metabolites above and N-butylthiocarbamic acid, with MBT as the penultimate and MBT sulfoxide as the ultimate inhibitor of mALDH.
    Chemical Research in Toxicology 05/1998; 11(5):535-43. DOI:10.1021/tx980002l · 4.19 Impact Factor
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    ABSTRACT: S-Methylation is a new bioactivation mechanism for metam and metabolites of methyl isothiocyanate and dazomet in mice. These soil fumigants are converted to S-methyl metam [MeNHC(S)SMe] which reaches peak levels in liver, kidney, brain, and blood 10-20 min after intraperitoneal (ip) treatment. The half-life of S-methyl metam administered ip is 8-12 min in each of these tissues. S-Methyl metam-oxon [MeNHC(O)SMe] is also detected as a metabolite of each of these soil fumigants on analysis by gas chromatography/mass spectrometry with chemical ionization. The conversion of methyl isothiocyanate to S-methyl metam and its oxon probably involves conjugation with glutathione, hydrolysis to S-(N-methylthiocarbamoyl)-cysteine, cleavage by cysteine conjugate beta-lyase to release metam, and finally methylation and oxidative desulfuration. Metam and dazomet are converted to S-methyl metam by mouse liver microsomes on fortification with S-adenosylmethionine. Metam, methyl isothiocyanate, dazomet, and three metabolites (metam-oxon [MeNHC(O)SH], MeNHC(S)SMe, and MeNHC-(O)SMe) administered ip to mice at 40 mg/kg inhibit low-Km liver mitochondrial aldehyde dehydrogenase and elevate ethanol-dependent blood and brain acetaldehyde levels. Several fungicides including the dialkyldithiocarbamates as the disulfide (thiram and the related alcohol-abuse drug disulfiram) and metal salts (ziram) also yield S-methyl thiocarbamate metabolites. Eight S-alkyl and S-(chloroallyl) thiocarbamate herbicides (EPTC, molinate, butylate, vernolate, pebulate, diallate, sulfallate, and triallate), but not their S-chlorobenzyl analog (thiobencarb), undergo sequential liberation of the thiocarbamic acid and then S-methylation, forming the S-methyl thiocarbamates which are new metabolites and potential aldehyde dehydrogenase inhibitors. The S-methyl mono- and dithiocarbamate metabolites of these herbicides and fungicides are easily identified by retention time on gas chromatography and by mass spectrometry giving [MH]+ plus [R1R2NCO]+ or [R1R2NCS]+, respectively, as the two major ions.
    Chemical Research in Toxicology 01/1996; 8(8):1063-9. DOI:10.1021/tx00050a010 · 4.19 Impact Factor
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    ABSTRACT: The metabolism of cycloate, a thiocarbamate herbicide, was investigated in mature radish leaf. Twelve new metabolites were identified by liquid chromatographic/mass spectrometric analysis using fast atom bombardment and packed capillary liquid chromatography columns. Full-scan and tandem mass spectrometric methods were employed. Application of the on-column focusing technique resulted in identifications with injections of as little as 15 ng of metabolite (20 ppb in radish). This injection technique allows the practical use of packed capillary liquid chromatography/mass spectrometry in sample-limited applications. Cycloate is oxidized to several ring-hydroxylated isomers that are subsequently glucosylated and esterified with malonic acid. Cycloate is also conjugated with glutathione. Metabolic hydrolysis of the glutathione conjugate formed a cysteine conjugate that is further metabolized by amidation with either malonic or acetic acid. Transamination of the cysteine conjugate gave a thiolactic acid derivative. Metabolites were also identified that were the result of both ring-hydroxylation and conjugation with glutathione. One of these, an N-acetylcysteine conjugate, is the first report of a mercapturic acid in plants. The structures of two of the new metabolites were confirmed by chemical synthesis.
    Biological Mass Spectrometry 10/1994; 23(10):626-36. DOI:10.1002/bms.1200231005

Publication Stats

82 Citations
22.33 Total Impact Points

Institutions

  • 1996–1999
    • University of California, Berkeley
      • Department of Environmental Science, Policy, and Management
      Berkeley, California, United States