Mechanism for Benomyl Action as a Mitochondrial Aldehyde Dehydrogenase Inhibitor in Mice

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.

Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease

“…2 B and D). These results are consistent with those of Staub et al for hepatic mitochondria prepared from mice (18). UPS Inhibition.…”

“…Microtubule inhibitors disrupt the ubiquitin-proteasome system (UPS) (17) and cause selective dopaminergic cell damage and aggregation of α-synuclein, the predominant component of an intracytosolic Lewy body, which is the pathologic hallmark of PD (16). Furthermore, benomyl inhibits aldehyde dehydrogenase (ALDH) activity in liver and brain mitochondria (18,19), although ALDH inhibition has not been measured directly in brains in vivo. The mitochondrial-associated ALDH2 is of particular interest because it metabolizes toxic aldehydes in brain tissue, including the dopamine (DA) metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) (Fig.…”

“…We previously showed that benomyl inhibits ALDH activity in vivo with indirect evidence in brains as elevated acetaldehyde levels upon ethanol challenge (18). The ALDH inhibitory activity is caused by BIC and its downstream metabolites including S-methyl N-butylthiocarbamate (MBT), which is further converted by cytochrome P450 (CYP) enzymes to MBT sulfoxide (MBT-SO), a very potent ALDH inhibitor (18). We have also shown that benomyl is an inhibitor of 26S UPS activity (24), but here we find that this occurs at micromolar concentrations.…”

Donor CD4+ Foxp3+ regulatory T cells are necessary for posttransplantation cyclophosphamide-mediated protection against GVHD in mice

“…However, mafosfamide treatment led to persistently higher ALDH2 expression by Tregs compared with Tcons at day 7 (supplemental Figure 6B). Furthermore, ALDH2 inhibition with the ALDH inhibitors DEAB 38 or Benomyl 39,40 led to markedly worse Treg survival after mafosfamide treatment (supplemental Figure 6C), thus linking ALDH2 expression to mafosfamide resistance in Tregs. Importantly, ALDH2 inhibition alone had no effect on Tcon viability, while Tcons in mafosfamidetreated cultures had significantly reduced viability, regardless of ALDH2 inhibition (supplemental Figure 6C).…”

Aldehyde dehydrogenase variation enhances effect of pesticides associated with Parkinson disease

“…Benomyl inhibits ALDH after being metabolized into thiocarbamate compounds. 36 Consistently, all of the coordinating dithiocarbamates inhibited ALDH activity, suggesting a common mechanism. The carbamates aldicarb and methomyl did not inhibit ALDH, so the thiol group is likely responsible for the inhibitory function of the dithiocarbamates, 37 although these compounds can also evolve carbon disulfide gas, which has been shown to cross-link proteins such as ALDH.…”