Mechanism for Benomyl Action as a Mitochondrial Aldehyde

and Management, 114 Wellman Hall, University of California, Berkeley, California 94720-3112 ..... CA) were administered the test compounds ip using wa...
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Chem. Res. Toxicol. 1998, 11, 535-543

535

Mechanism for Benomyl Action as a Mitochondrial Aldehyde Dehydrogenase Inhibitor in Mice Richard E. Staub, Gary B. Quistad, and John E. Casida* Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 114 Wellman Hall, University of California, Berkeley, California 94720-3112 Received January 5, 1998

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 alcoholsensitizing 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-(Nbutylcarbamoyl) conjugates. Benomyl and MBT inhibit mouse hepatic mALDH in vitro with IC50s of 0.77 and 8.7 µM, 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 µM, 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 µM). 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.

Introduction Fungicidal dimethyldithiocarbamates, as salts and disulfides such as thiram [Me2NC(S)SSC(S)NMe2], have long been of interest as sensitizers to ethanol and inhibitors of hepatic mitochondrial low-Km aldehyde dehydrogenase (mALDH or ALDH2)1 activity (1, 2). The thiram homologue disulfiram [Et2NC(S)SSC(S)NEt2] is the only available drug used in the aversion therapy of recovering alcoholics (3). Other pesticidal mALDH inhibitors are fumigants such as metam [MeNHC(S)SNa] (4) and herbicides such as S-ethyl N,N-dipropylthiocarbamate [EPTC; Pr2NC(O)SEt] (5, 6). These compounds * To whom correspondence should be addressed. Tel: 510-642-5424. Fax: 510-642-6497. E-mail: [email protected]. 1 Abbreviations: ALDH, aldehyde dehydrogenase; BIC, butyl isocyanate; CySBT, S-(N-butylcarbamoyl)cysteine; CySH, cysteine; EPTC, S-ethyl N,N-dipropylthiocarbamate; GSBT, S-(N-butylcarbamoyl)GSH; IC50, concentration or dose for 50% inhibition; mALDH, mitochondrial aldehyde dehydrogenase (ALDH2, mouse hepatic low-Km in this study); MBT, S-methyl N-butylthiocarbamate; MBT-SO, S-methyl N-butylthiocarbamate sulfoxide; MMPP, magnesium monoperoxyphthalate; NAcCySBT, N-acetyl-S-(N-butylcarbamoyl)cysteine; NAcCySH, Nacetylcysteine; NBI, N-benzylimidazole; N-OH-MBT, S-methyl N-butylN-hydroxythiocarbamate; P450, cytochrome P450; PB, piperonyl butoxide; SAM, S-adenosylmethionine; SIM, selected ion monitoring; Ac, acetyl; Bu, n-butyl; Et, ethyl; Me, methyl; Pr, n-propyl; Ph, phenyl.

are all di- or monothiocarbamates, and it was therefore surprising to find in a survey of fungicides that benomyl, which does not contain a thiol functional group (Figure 1), has similar potency as an in vivo mALDH inhibitor. The mechanism by which benomyl inhibits mALDH might be analogous to that known for the thiocarbamate pesticides indicated above if they proceed through common metabolic pathways leading to formation of S-alkyl thiocarbamates and/or the corresponding sulfoxides. Previous studies suggest this might be the case, since benomyl is metabolized in rats to S-(N-butylcarbamoyl)GSH (GSBT) evident in bile (7) and S-(N-butylcarbamoyl)cysteine (CySBT) and N-acetyl-S-(N-butylcarbamoyl)cysteine (NAcSBT) identified in urine (8). GSBT and CySBT are analogous to the corresponding conjugates of methyl isocyanate (9-11), thereby potentially serving as latent releasers of butyl isocyanate (BIC) in vivo (7, 10, 12). The GSH and cysteine (CySH) conjugates also provide a potential route to S-methyl N-butylthiocarbamate (MBT) as an activation product through β-lyase cleavage of CySBT and subsequent S-methylation as described for methyl isothiocyanate and methyl isocyanate (4).

S0893-228x(98)00002-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/23/1998

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Staub et al.

Figure 1. Proposed pathways for bioactivation of benomyl analogues and metabolites as inhibitors of mALDH. Benomyl, BIC, GSBT, CySBT, NAcCySBT, and MBT are also candidate direct inhibitors of mALDH.

In the current investigation, we find that benomyl is metabolized to MBT, which then undergoes further metabolic activation. The bioactivation of dialkylthiocarbamates as mALDH inhibitors involves conversion to the sulfoxides (5, 6, 13). Alternatively, O-ethyl Nhydroxycarbamate is proposed as a possible bioactivation product of ethyl carbamate (14). The sulfoxide or Nhydroxy derivative is not known for any monoalkylthiocarbamate despite the obvious potential relevance of these compounds in metabolism studies. This report establishes the mechanism of benomyl bioactivation in mice relative to its metabolic conversion to MBT and further activation in vitro and in vivo to the most potent pesticide-derived inhibitor known for mALDH (Figure 1).

Materials and Methods Chromatography and Spectroscopy. Reversed-phase HPLC was performed using a Hewlett-Packard 1050 solvent delivery system and 1040M Series II photodiode array detector monitoring at 220 nm. For purity analysis, an Ultremex 5 C18 column (Phenomenex, Rancho Palos Verdes, CA) (5 µm, 0.46 × 25 cm) was employed with a linear gradient of 0-50% methanol in water with constant 0.1% trifluoroacetic acid over 20 min and finally a linear gradient to 100% methanol over an additional 10 min, each at 1 mL/min; tR (min) MBT 21.3, GSBT 16.2, CySBT 13.9, and NAcSBT 17.1. For radiopurity analysis of [14C]MBT and to examine conjugates as metabolites, a Vydac C4 protein column (Separations Group, Hesperia, CA) (5 µm, 0.46 × 25 cm) was used with acetonitrile in water at constant 0.1% trifluoroacetic acid, with an initial 0% acetonitrile for 5 min followed by linear gradients of 0-40% acetonitrile over 15 min, 40-60% over 10 min, and then 60% for 10 min, all at 1.5 mL/ min; tR (min) MBT 17.2, GSBT 12.8, and NAcCySBT 15.1. The eluent was monitored at 220 nm, and when appropriate 1-min fractions were taken for liquid scintillation counting. NMR spectra were recorded with a Bruker WM-300 spectrometer at 300 MHz for 1H and 75 mHz for 13C in solvents as specified. Chemical shifts (ppm) for 1H NMR were referenced to the solvent peak for CDCl3 and to tetramethylsilane for D2O solutions, and for 13C NMR the reference was methanol for D2O solutions. GC/MS with chemical ionization using CH4 was performed on a Hewlett-Packard 5890 gas chromatograph connected to a 5971A mass spectrometer: HP-1701 (14% CNPrPh Me siloxane) fused silica gel capillary column (30-m × 0.25-mm i.d.) (HewlettPackard, Wilmington, DE); 70-250 °C over 20 min. For

identification of metabolites, the scan mode was used monitoring all ions between m/z 50 and 500. Quantitative analysis involved a 1.0-µL aliquot introduced with the HP7673A automatic sample injector using selected ion monitoring (SIM). Chemicals. (A) General. Benomyl and carbendazim were obtained from Chem Service (West Chester, PA), thiophanatemethyl was from Elf Atochem North America, Inc. (King of Prussia, PA), and BIC was from Eastman Kodak Co. (Rochester, NY). EPTC, N-depropyl EPTC [PrHNC(O)SEt], and S-methyl EPTC [Pr2NC(O)SMe] were available from previous studies in this laboratory. S-Methyl EPTC sulfoxide and sulfone were prepared by oxidation of S-methyl EPTC with magnesium monoperoxyphthalate (MMPP) in D2O/methanol (discussed later) and m-chloroperoxybenzoic acid in chloroform (15), respectively. The sources for other chemicals were N-benzylimidazole (NBI), MMPP (80% pure), and methimazole (2-mercapto1-methylimidazole) from Aldrich Chemical Co. (St. Louis, MO); [1-14C]-n-butylamine hydrochloride from American Radiolabeled Chemicals, Inc. (St. Louis, MO); aldehyde dehydrogenase (ALDH) purified from yeast (20-40 units/mg of protein) and aminooxyacetic acid from Sigma (St. Louis, MO). (B) MBT, [methyl-13C]MBT, and [butyl-14C]MBT. The MBT intermediate, N-butylthiocarbamic acid, was synthesized by bubbling carbonyl sulfide for 3 h into a solution of nbutylamine (5 g) in ethanol (25 mL) by the general procedure of Johansson et al. (16). MBT itself was obtained by treatment of the thioic acid with equimolar iodomethane in acetone saturated with potassium carbonate for 30 min at 25 °C. Similarly, [methyl-13C]MBT was produced by reaction with [13C]iodomethane (Aldrich). The residue from evaporation of the acetone was resuspended in dichloromethane (50 mL), partitioned with water and purified by flash chromatography (silica gel 60, eluted with hexane/acetone, 7:3). Combined fractions were diluted to approximately 200 mL with hexane, and MBT crystallized at -20 °C as white needles: mp 34 °C; 1H NMR (CDCl3) δ 0.92 (t, 3H, CH3), 1.36 (m, 2H, CH2), 1.50 (m, 2H, CH2), 2.34 (s, 3H, SCH3), 3.29 (m, 2H, CH2), 5.31 (br s, 1H, NH); 13C NMR (CDCl ) δ 11.65 (s, CH ); purity > 95% based on GC/ 3 3 MS and HPLC as above. [butyl-14C]MBT was synthesized by a procedure based on Tilles and Antognini (17) involving the coupling of [1-14C]-n-butylamine hydrochloride (100 µg) (55 mCi/ mmol in 0.5 mL of ethanol) to methyl chlorothioformate (2 molar equiv) in ether (3 mL) with triethylamine (1.1 molar equiv). The reaction mixture was stirred at room temperature for 15 min before partitioning with water (1 mL). The ether phase was concentrated, and [14C]MBT was purified by preparative C18 HPLC (radiopurity by C4 HPLC > 96%).

Benomyl Inhibition of Aldehyde Dehydrogenase (C) S-(N-Butylthiocarbamoyl) Conjugates. GSBT was prepared by the method of Han et al. (18) involving addition of BIC (120 mg) in acetone (2 mL) to GSH (185 mg) in acetonitrile/ water (7:3) (10 mL), stirring for 30 min at 25 °C, filtering, and washing the conjugate with acetone. NAcCySBT was prepared using the procedure of Hubbell and Casida (19) in which BIC (100 µL) was added to N-acetylcysteine (NAcCySH) (100 mg) in methanol (15 mL) and triethylamine (5 mL) followed by stirring for 18 h at 25 °C and workup as above. CySBT was prepared by addition of BIC (650 µL) to CySH (250 mg of free acid) in acetonitrile (25 mL) at 0 °C and then stirring the reaction mixture for 70 h at 25 °C under a drying column. The conjugates were subjected to preparative HPLC to obtain a purity of >90%, and structures were verified by fast atom bombardment mass spectrometry. (D) N-OH-MBT. S-Methyl N-butyl-N-hydroxythiocarbamate (N-OH-MBT) was prepared from n-butylhydroxylamine generated by converting ethyl 5-hydroxy-3-methyl-4-isoxazolecarboxylate sodium salt (Aldrich) to the N-butyl derivative (20) and then acid hydrolysis [water/glacial acetic acid/12 M hydrochloric acid (1:1:1), reflux, 16 h]. n-Butylhydroxylamine (oxalate salt, 4.5 mmol) was thoroughly dried and then stirred for 18 h with triethylamine (8.9 mmol) in ether (50 mL). Hexamethyldisilazane (Aldrich) (4.4 mmol) was injected through a septum and the reaction mixture under nitrogen stirred for an additional 24 h at 25 °C (21). Methyl chlorothioformate (Aldrich) (4 mmol) was then added and allowed to react for 12 h at 25 °C to form N-(O-trimethylsilyl)-N-butylthiocarbamate: 1H NMR (CDCl3) δ 0.26 (s, 9H, trimethylsilyl), 0.91 (t, 3H, CH3), 1.31 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.24 (s, 3H, SCH3), 3.60 (t, 2H, CH2). The silyl protecting group was removed with methanol (20 min) to give N-OH-MBT: 1H NMR (CDCl3) δ 0.93 (t, 3H, CH3), 1.33 (m, 2H, CH2), 1.63 (m, 2H, CH2), 2.27 (s, 3H, SCH3), 3.63 (t, 2H, CH2); verified by GC/MS analysis of the O-acetyl derivative (tR ) 10.4 min, [M + 1]+ ) m/z 206, [BuN(OAc)CO]+ ) m/z 158), synthesized from the reaction of acetic anhydride with N-OHMBT in dry ether over solid potassium carbonate. N-OH-MBT is stable in methanol, 100 mM phosphate buffer (pH 7.4), and 50 mM pyrophosphate buffer (pH 9.0) as no decomposition was detected by 1H NMR after 24 h. (E) MBT-SO. To prepare MBT sulfoxide (MBT-SO) for 1H NMR characterization, a solution of MBT (3 mg) in methanold4 (50 µL) and D2O (350 µL) was cooled in an ice bath, and then MMPP (16.5 µmol, 1.6 peracid equiv) was added in D2O (60 µL). The reaction was also carried out with [methyl-13C]MBT added in methanol for 13C NMR monitoring. Treatment of MBT with MMPP results in a downfield shift of the S-methyl 13C and 1H resonances consistent with the generation of the sulfoxide moiety (15), i.e., the NMR chemical shifts are similar for the oxidation products of MBT and S-methyl EPTC in D2O (Table 1). MBT sulfone and S-methyl EPTC sulfone could not be generated using MMPP oxidation in D2O/methanol-d4 as above, and MBT-SO was not observed among the complex mixture of oxidation products of MBT with m-chloroperoxybenzoic acid in CDCl3. MBT-SO was characterized as to hydrolytic stability and reactions with thiol compounds. To calculate the half-life of MBT-SO in 100 mM phosphate buffer (pH 7.4), [methyl-13C]MBT (3 mg) was oxidized with 1.6 equiv of MMPP at 22 °C as above followed by 13C NMR monitoring every 5 min of the area of the MBT-SO resonance relative to that of methanol as an internal standard. In the reaction of MBT-SO to form S-(N-butylcarbamoyl) conjugates, MBT (0.25-1.5 mg) in methanol (25 µL) was added to cold water (200 µL) or 100 mM phosphate buffer (pH 7.4) (100 µL), then MMPP (1.6 equiv) in water (20 µL) was added, and the mixture was briefly shaken. Excess NAcCySH (18 µmol) or GSH (16 µmol) was immediately added, and the reaction was allowed to proceed for 20 min at room temperature before C4 HPLC analysis. Mice and Treatment Protocols. Male albino SwissWebster mice (24-34 g) from Simonsen Laboratories (Gilroy, CA) were administered the test compounds ip using water (for

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 537 Table 1. 13C and 1H NMR Chemical Shifts for the S-Methyl Substituents of Two Thiocarbamates and Their Sulfoxide and Sulfone Derivatives NMR chemical shift of S(On)Me moiety ppm compound (solvent)

S

SO 13C

difference SO2

SO-S SO2-SO SO2-S

NMR

Pr2NC(O)S(On)Me CDCl3a 13.37 37.59 40.23 24.22 D2O 10.49 36.24b 39.26c,d 25.75 BuHNC(O)S(On)Me D2O 11.65 36.80b NDd 25.15 1H

Pr2NC(O)S(On)Me CDCl3a D2O BuHNC(O)S(On)Me D2O a

2.64 3.02

26.86 28.77

0.32 0.44

0.83 0.93

NMR

2.31 2.29

2.82 3.14 2.78b 3.22c,d

0.51 0.49

2.30

2.84b NDd

0.54

b

Data from Wu et al.. (15). NMR shift determined for product from oxidation with MMPP. Methanol or methanol-d4 was used at 25% to solubilize Pr2NC(O)SMe. c NMR shift determined on product from oxidation with m-chloroperoxybenzoic acid. d No RS(O2)Me detected following MMPP oxidation.

conjugates) or Me2SO (for the other compounds) as the carrier vehicle. Additionally, benomyl was dosed orally using corn oil (300 µL). At appropriate times the animals were sacrificed by cervical dislocation and immediately dissected to obtain the liver. MBT Analysis, Enzymatic Formation, and Metabolism. Identification of MBT in treated mice involved homogenizing the liver in water with metabolite recovery on partitioning into dichloromethane. The dichloromethane phase was dried (anhydrous sodium sulfate) and evaporated to ca. 0.1 mL before GC/MS analysis in the scan mode. For quantitation of MBT, fresh samples of liver (1.3-2.1 g) from individual mice were homogenized in water (2 mL) which was then partitioned twice with dichloromethane (2 × 2 mL) containing N-depropyl EPTC as the internal standard (0.1 µg/mL). A 1-µL aliquot of the dichloromethane extract was analyzed by GC/MS-SIM, monitoring MH+ at m/z 148 and the isocyanate fragment ion, [BuNHCO]+, at m/z 100. Quantitation of MBT as a metabolite involved comparison to a standard curve for the authentic compound, corrected for recovery of the internal standard. For enzymatic production of MBT, CySBT (0.9 mM) was incubated with S-adenosylmethionine (SAM) (1.1 mM) and mouse liver microsomes (1 mg of protein) in 100 mM phosphate buffer (pH 7.4) (0.5 mL) for 60 min at 37 °C; then a dichloromethane (1 mL) extract was analyzed by GC/MS-SIM as above. Hydroxylamine and aminooxyacetic acid (1 mM each) were added to specific assays as inhibitors of pyridoxal-requiring enzymes (e.g., β-lyase) (22, 23). For controls, SAM was not added or heat-denatured microsomes (80 °C, 10 min) were used. The metabolism of MBT was examined in mice treated ip with [butyl-14C]MBT (27 mCi/mmol) (0.01 mmol/kg). The 0-2-h urine was analyzed directly by C4 HPLC and by two-dimensional TLC on silica gel 60 (0.5-mm thickness, 20- × 20-cm plates) developed in both directions with ethyl acetate/methanol/water (14:6:1). For cochromatography, NAcCySBT (100 µg) was mixed with the urine sample, detecting the standard (Rf 0.38) as a brownishyellow spot when sprayed with palladium chloride (0.5 mg/mL in 50% aqueous ethanol) and the radiolabeled metabolite(s) with a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Possible formation of N-OH-MBT was examined by incubating MBT (25 µg) with microsomes (3 mg) and NADPH (1 mg) in 100 mM phosphate buffer (pH 7.4) (1 mL) for 10 min at 37 °C and then analysis of an ether extract (1 mL) by drying (sodium sulfate), acetylation, and GC/MS as above. ALDH Activity and Inhibition Assays. Mouse hepatic mALDH was assayed by a described procedure (5) modified only in using 250 µg of protein/assay and replacing 0.25 M sucrose

538 Chem. Res. Toxicol., Vol. 11, No. 5, 1998 throughout with 0.25 M sucrose containing 0.5 mM EDTA and 5 mM Tris (pH 7.2). In vitro inhibition studies involved incubation of the inhibitor and enzyme for 10 min at 25 °C before substrate addition. For in vivo inhibition studies, the test compounds were administered ip (benomyl also dosed orally), at 0.0003-0.27 mmol/kg, followed after 2 h by sacrifice and assay of mALDH activity (250 µg of protein/assay) (5). Additionally, treated mice were sacrificed at extended times to measure the persistence of mALDH inhibition from a single dose. Controls received carrier solvent only. The figures in this report are plotted from composite data for all experiments, whereas the tables give the mean and SE values from individual experiments, and as such there are sometimes small differences in the IC50s in the figures and tables. Inhibition of yeast ALDH was determined by incubating 0.125 IU of purified enzyme with the inhibitor in 50 mM pyrophosphate buffer (pH 9.0) (1 mL) with NAD (50 mM). After 10 min, acetaldehyde (250 µM) was added, and the conversion of NAD to NADH was monitored at 340 nm for 5 min. Acetaldehyde levels in blood and brain of mice were determined as the O-benzyloxime ether 30 min after two ip treatments, the first with benomyl at 160 mg/kg and 2 h later with ethanol at 1000 mg/kg (5). MBT-SO, because of its instability, was generated immediately before mALDH and yeast ALDH inhibition assays by adding MBT (41 mM) to 1.6 equiv of MMPP in ice-cold water (250 µL). After mixing, serial dilutions were immediately made in cold 50 mM pyrophosphate buffer (pH 9.0) for assay of ALDH inhibition. The mALDH assay was performed as above except the mitochondria were first disrupted by osmotic dilution (addition of distilled water) and freezing before removal of the membranes by centrifugation, thereby potentially providing better interaction of the unstable inhibitor with the enzyme. Bioactivation of ALDH Inhibitors. Potential metabolic activation was measured by incubating hepatic mitochondrial protein (2.5 mg) with mouse liver microsomes (2-3 mg), NADPH (1 mg), and the candidate proinhibitor in 100 mM phosphate buffer (pH 7.4)/0.25 M sucrose (3 mL) for 10 min at 37 °C. After incubation, the mitochondria were isolated by centrifugation (4200g, 10 min), and the ALDH activity was assayed as above. For controls, NADPH or microsomes were deleted or a poor substrate (CySBT) was used. Candidate inhibitors of bioactivation were assayed with a 10-min preincubation before adding MBT as follows: for cytochrome P450 (P450) oxidasessNBI (24), piperonyl butoxide (PB) (25), R-naphthoflavone (26), sulfaphenazole (26), quinidine (26), and 4-(2-thiazolylazo)orcinol (26); for flavin-containing monooxygenasessmethimazole (27). To determine the extent of microsomal oxidation of MBT (0.034-1.7 µM), the above reaction was run with and without microsomes and NADPH. Instead of isolating the mitochondria for mALDH assay, the reaction mixture was extracted with dichloromethane (1 mL) for analysis of unmetabolized MBT by GC/MS-SIM. Possible regeneration of mALDH activity inhibited in vitro by MBT or EPTC with metabolic activation was determined by preparing inhibitor-bound mouse hepatic mALDH. The mitochondria were isolated from the microsomes and NADPH by centrifugation (4200g, 10 min), resuspended in fresh buffer with phenylmethanesulfonyl fluoride (100 µM), and assayed for ALDH activity over 6 h (24 °C). The experiment was also run in the presence of 5 mM GSH.

Results Benomyl Inhibits Hepatic Low-Km mALDH and Ethanol Detoxification in Vivo. It was anticipated in a survey of fungicides and herbicides that activity would be restricted to di- and monothiocarbamates. It was therefore surprising to observe that benomyl administered ip to mice inhibits mALDH activity with an IC50 of 0.024 mmol/kg (7.0 mg/kg) (Figure 2) and is almost equipotent following oral treatment (Table 2). Thiopha-

Staub et al.

Figure 2. Benomyl and two metabolites as in vivo inhibitors of mouse hepatic low-Km mALDH. Mice were sacrificed 2 h after ip treatment. Values are based on 3-8 replicates with error bars showing SE. Table 2. Benomyl and Metabolites as in Vivo Inhibitors of Mouse Hepatic Low-Km mALDH inhibition (% ( SE)a compound

0.27 mmol/kg

0.03 mmol/kg

benomyl carbendazimc BIC GSBT CySBT NAcCySBT MBT

90 ( 16, 10 88 ( 3 88, 90 78, 62 66, 76 94 ( 1

54 ( 14b 3.0 ( 4.1 43 ( 21 19, 16 2, 13 26, 13 90 ( 3

5b

a 2 h after ip administration. Mean ( SE based on 3-8 experiments or individual values with 2 experiments. b Comparable inhibition values after oral administration were 88% and 91% at 0.27 mmol/kg and 34% and 36% at 0.03 mmol/kg. c The fungicide thiophanate-methyl gives no inhibition at 0.27 mmol/ kg.

Table 3. Benomyl and Metabolites as in Vitro Inhibitors of Mouse Hepatic Low-Km mALDH compound

IC50 (µM ( SE)a

benomyl carbendazimc BIC GSBT CySBT NAcCySBT MBTf N-OH-MBT MBT-SO

0.77b >250 (3 ( 4%)d 1.2 ( 0.2 0.65 ( 0.06 0.55 ( 0.07e 1.8 ( 0.2 9.3 ( 1.0 52 ( 17 0.090 ( 0.011

a Mean ( SE based on 3 experiments. b Average of 2 experiments differing by 1000-fold less active than benomyl in vitro as mALDH inhibitors, whereas N-depropyl EPTC is of moderate potency (IC50 of ∼40 µM) (Table 3).

Benomyl Inhibition of Aldehyde Dehydrogenase

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 539

Figure 4. MBT identification as a metabolite after 120 min in the liver of benomyl-treated mice (100 mg/kg ip) based on GC/ MS comparison (tR and fragmentation) with an authentic standard. The upper trace depicts the total ion chromatogram, while the lower trace shows the parent ion (M + 1) and fragment ions detected for the MBT peak at 8.78 min.

Figure 3. Benomyl and three metabolites as in vitro inhibitors of mouse hepatic low-Km mALDH and yeast ALDH activities. The inhibitors were incubated with the mouse mitochondrial preparation (250 µg of protein) or purified yeast ALDH (0.125 IU) for 10 min at 25 °C prior to adding the acetaldehyde substrate (50 and 250 µM, respectively). Values are means for 2 replicates differing by