Characterization of S-(N, N-Dialkylaminocarbonyl) cysteine Adducts

rat globin analogous to that reported for disulfiram and to inhibit aldehyde ... of molinate to act as a peripheral demyelinating agent similar to dis...
0 downloads 0 Views 145KB Size
Download PDF
258

Chem. Res. Toxicol. 2004, 17, 258-267

Characterization of S-(N,N-Dialkylaminocarbonyl)cysteine Adducts and Enzyme Inhibition Produced by Thiocarbamate Herbicides in the Rat Lisa J. Zimmerman, Holly L. Valentine, and William M. Valentine* Department of Pathology and Center in Molecular Toxicology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2561 Received October 14, 2003

Thiocarbamates are a major class of herbicides used extensively in the agricultural industry. It has been shown that thiocarbamates can form reactive sulfoxide and sulfone intermediates, which may be involved in the toxicity of thiocarbamates through covalent modification of cysteine and serine active sites of enzymes. Molinate has been shown to generate an S-hexahydro-1H-azepine-1-carbonyl adduct on the Cys-125 residue of the β2- and β3-chains of rat globin analogous to that reported for disulfiram and to inhibit aldehyde dehydrogenase and nonspecific esterase activity. The present study examined whether other thiocarbamate herbicides produce similar covalent protein modifications and enzyme inhibition to that reported for molinate and whether S-(N,N-dialkylaminocarbonyl)cysteine adduct levels are correlated to enzyme inhibition or the structure of thiocarbamate herbicides. Additionally, the potential of molinate to act as a peripheral demyelinating agent similar to disulfiram was evaluated. To address these aims, rats were exposed ip to molinate, vernolate, ethiolate, EPTC, or butylate for 5 days after which hemogloblin was isolated and analyzed for protein adducts using HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In addition, brain, liver, and testes mitochondrial and microsomal fractions were assayed for nonspecific esterase, low Km ALDH, or total ALDH activities, and S-(N,N-dialkylaminocarbonyl)cysteine adducts were measured by LC/MS/MS. For the neurotoxicity assessments, rats were administered molinate parenterally for subchronic periods and morphological evaluations performed on peripheral nerves. All of the thiocarbamates except butylate produced S-(N,N-dialkylaminocarbonyl)cysteine adducts on globin and the quantity of adducts detected decreased with increasing size of the nitrogen substituents. In contrast, a clear relationship between cysteine modification in mitochondrial and microsomal samples to nitrogen substituents was not evident, and although molinate produced relatively high levels of adducts and esterase inhibition and butylate low levels of adducts and esterase inhibition for most samples, in general, the level of S-(N,N-dialkylaminocarbonyl)cysteine adducts did not appear to be related to enzyme inhibition. Molinate did not produce segmental demyelination in peripheral nerve, suggesting that molinate and possibly other thiocarbamates do not share the neurotoxic potential of dithiocarbamates.

Introduction Thiocarbamates are a major class of herbicides used extensively in the agricultural industry. It has been shown that thiocarbamates undergo metabolic bioactivation via S-oxidation to form reactive sulfoxide and sulfone intermediates, which may be involved in the toxicity of thiocarbamates observed in mammals and fish (1-3). Molinate (1a), a selective herbicide used in the rice industry, has been found to produce male reproductive toxicity in rats and has also been shown to be highly hepatotoxic to the common carp (4, 5). Previous studies have implicated metabolic bioactivation via S-oxidation of molinate to the reactive metabolites molinate sulfone (2a) and molinate sulfoxide (4a) to be involved in the molinate-induced testicular toxicity observed in the rat * To whom correspondence should be addressed. Tel: 615-343-5836. Fax: 615-343-9825. E-mail: [email protected].

following administration. The mechanism of testicular toxicity induced by molinate is proposed to involve the inhibition of hydrolase A (6, 7), a carboxylesterase required in the biosynthesis of testosterone, presumably via modification of an active site serine residue by molinate or one of its metabolites. A similar bioactivation sequence via S-oxygenation has been reported for other thiocarbamate herbicides, such as thiobencarb and EPTC1 (1d), and it has been suggested that the toxicity associ1 Abbreviations: ACN, acetonitrile; ALDH, aldehyde dehydrogenase; BUT-Cys, S-(N,N-diisobutylaminocarbonyl)cysteine; m-CPBA, 3-chloroperoxybenzoic acid; CID, collision-induced dissociation; COS, carbonyl sulfide; EPTC-Cys, S-(N,N-dipropylaminocarbonyl)cysteine; ESI, electrospray ionization; ETH-Cys, S-(N,N-diethylaminocarbonyl)cysteine; HHAC-Cys, S-(hexahydro-1H-azepinecarbonyl)cysteine; MALDITOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; NAD+, nicotinamide adenosine dinucleotide (oxidized form); PIP-Cys, S-(piperidin-1-ylcarbonyl)cysteine; SA, sinapinic acid; SRM, selected reaction monitoring; TFA, trifluoroacetic acid; VER-Cys, S-(N,N-dipropylaminocarbonyl)cysteine.

10.1021/tx034209c CCC: $27.50 © 2004 American Chemical Society Published on Web 01/23/2004

Thiocarbamate Adducts and Enzyme Inhibition

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 259 Scheme 1

ated with such compounds is the result of sulfoxidation to reactive intermediates, possibly leading to an increase in their hepatotoxic potential (1, 8). In addition to sharing a similar metabolism with molinate, the structurally similar thiocarbamates vernolate (1b), ethiolate (1c), EPTC, and butylate (1e) have also been shown to be potent inhibitors of low Km ALDH in vivo in the rat, which is thought to occur through the formation of their respective sulfoxide and/or sulfone (Scheme 1) (9-11). Molinate has recently been shown to covalently modifiy rat hemoglobin in vivo in a cumulative dose-dependent manner through the formation of a S-(hexahydro-1Hazepine-1-carbonyl) adduct (10a) on the Cys-125 residue of the β2- and β3-chains (12). The covalent modification produced by molinate is consistent with the proposed bioactivation of the parent compound to a reactive electrophilic sulfoxide or sulfone intermediate capable of reacting with sulfhydryl groups on proteins. These modifications are analogous to those identified for disulfiram, a dithiocarbamate, used clinically to treat alcoholism, that acts through inhibition of ALDH (13). Disulfiram has been shown to carbamylate proteins in vivo, and carbamylation of an active site cysteine residue of mitochondrial ALDH is proposed as the mechanism for the observed inhibition of this enzyme (14). Disulfiram has also been shown to produce a peripheral neuropathy that targets Schwann cells resulting in demyelination (15). If protein carbamylation is a contributing process in disulfiram neurotoxicity, then molinate and other thiocarbamate pesticides may also be neurotoxic through a similar mechanism and may be more potent neurotoxicants as a result of the fewer bioactivation steps required to generate a reactive sulfoxide or sulfone. The current investigation was performed to determine whether in vivo exposure to vernolate, ethiolate, EPTC, and butylate results in similar covalent protein modifications to those produced by molinate. The effects of these thiocarbamates on plasma and tissue esterase and ALDH activities in the rat were assessed to determine if adduct levels were correlated to enzyme inhibition and to investigate the influence of nitrogen and sulfur substit-

uents upon the distribution and bioactivation of thiocarbamate herbicides. Additionally, the potential of molinate to act as a peripheral demyelinating agent analogous to disulfiram was evaluated. To address these aims, rats were exposed to molinate, vernolate, ethiolate, EPTC, or butylate for a period of 5 days after which time hemogloblin was isolated and analyzed for protein adducts using HPLC and mass spectrometry. In addition, plasma, brain, liver, and testes mitochondrial and microsomal fractions were assayed for nonspecific esterase and low Km ALDH or total ALDH activities. For the neurotoxicity assessments, rats were administered molinate parenterally for subchronic periods and morphological evaluations were performed on the sciatic, posterior tibial, and muscular branch of the posterior tibial nerves. The results were evaluated to determine whether a relationship could be identified between the level of covalent protein modifications produced by each of the thiocarbamates and the enzyme activity observed following administration of these compounds and to determine the potential of molinate to produce a Schwannopathy. Such correlations may provide a basis for the development of biomarkers of exposure and effect and insight into the role of covalent protein modifications for thiocarbamatemediated enzyme inhibition and provide structureactivity relationships useful for predicting the relative toxicity of individual herbicides within the thiocarbamate class.

Materials and Methods Chemicals. Corn oil, NAD+, and p-nitrophenylacetate were purchased from Sigma Chemical Co. (St. Louis, MO). Dipropylamine, diisobutylamine, diethylamine, iodopropane, and iodoethane were purchased from Aldrich (St. Louis, MO). All other chemicals were obtained from other commercial sources. Animals and Thiocarbamate Exposures. This study was performed in accordance with the National Institutes of Health’s Guide for Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats, 225-250 g, obtained from Harlan (Indianapolis, IN), were housed in a room on a 12 h diurnal

260

Chem. Res. Toxicol., Vol. 17, No. 2, 2004

light cycle and given rodent chow and water ad libitum during a 5 day acclimation period. Body weights were determined prior to the admininistration of the initial dose of the thiocarbamates and then daily throughout the dosing regimen up to the time of euthanasia. Treated rats (n ) 4) were administered an intraperitoneal injection of 100 mg/kg daily of molinate, vernolate, ethiolate, EPTC, or butylate in corn oil for a period of 5 days. Control animals were treated with equivalent volumes of corn oil using an identical dosing regimen. On day 6 under anesthesia, the rats were euthanized by exsanguination via cardiac puncture within 20-24 h of administering the final dose of the thiocarbamates and tissues were collected. Brain, liver, blood, and testes were obtained based upon their potential as target organs for toxicity and to help characterize the ability of these compounds to distribute into the nervous system and testes. Globin Isolation. Following exsanguination by cardiac puncture, blood collected in a heparinized syringe was centrifuged to separate the plasma from the red cells. A 5 mM phosphate-buffered saline solution (pH 7.4) containing 150 mM NaCl was added in equal volume to the red cells, resuspended, and centrifuged at 3000g. The supernatant and the buffy coat were discarded, and the washing procedure was repeated twice more. The washed red cells were then lysed with a 2× volume of 5 mM phosphate buffer (pH 7.4) and centrifuged at 20 000g for 25 min. The hemolysate (supernatant) was removed and mixed with 100 µL of 1 M ascorbic acid and added dropwise to 10 mL of cold 2.5% oxalic acid in acetone and allowed to precipitate for 15 min. The mixture was then centrifuged at 12 000g for 10 min, the supernatant was aspirated, and the globin was washed by adding 5 mL of acetone, resuspended with a metal spatula, and centrifuged at 12 000g for 10 min. The supernatant was aspirated, and the globin pellet was dried under a stream of nitrogen. Plasma and the globin pellets were stored at -80 °C until analysis. Preparation of Subcellular Fractions. Brain, liver, and testes were immediately removed and placed in ice-cold buffer. Brains were placed in ice-cold 0.25 mM sucrose containing 10 mM Tris and 0.5 mM EDTA (pH 7.4) and homogenzied, and mitochondria were isolated according to the method of Petterson (16). The livers were placed in ice-cold buffer containing 0.25 mM sucrose, 10 mM Tris-HCl, and 0.5 mM EDTA (pH 7.2) and homogenized. The mitochondria and microsomes were then isolated using differential centrifugation (17, 18). Testes were placed in cold 1.15% KCl and 50 mM Tris-HCl (pH 7.4) and decapsulated, and the mitochondria and microsomes were isolated according to Feingold (19). All mitochodria and microsomes were stored at -80 °C until used. Synthesis of Thiocarbamates. The general procedure for the synthesis of thiocarbamates 1a-f is based on the following method as described for molinate(S-ethyl-hexahydro-1H-azepine1-carbothioate) (1a). Hexamethyleneimine (1.13 mL, 10 mmol), ethanol (10 mL), and 10 N NaOH (1 mL) were taken up in a 25 mL two-neck flask and fitted with a Dewar condenser containing dry ice and 2-propanol. The flask was stirred and cooled in a dry ice/2-propanol bath (-70 °C), and COS (1 mL) was condensed into the flask. The second neck was stoppered, and the temperature of the bath was increased to 0-10 °C and maintained in that temperature while keeping the Dewar condenser cold. COS evaporated and condensed into the reaction. After 4 h, the condenser was removed and iodoethane (0.88 mL, 11 mmol) was added with stirring for 2 h after which the EtOH was removed, and the residue was distributed between the water (10 mL) and the chloroform (20 mL) layers. The organic layer was separated and dried, and the solvent was removed using a rotary evaporator. The purity was >98% as determined by GC. 1H NMR (CDCl3): δ 1.27 (t, 3H, CH3), 1.51 (m, 4H, CH2), 1.70 (m, 4H, CH2), 2.88 (q, 2H, CH2CH3), 3.41 (t, 2H, ring CH2N), 3.52 (t, 2H, ring CH2-N). 13C NMR (CDCl3): δ 15.8 (CH3), 24.9 (CH2CH3), 27.4 and 27.6 (CH2), 28.3 and 28.8 (CH2), 47.6 and 48.0 (CH2N), 168.2 (CdO). Spectral and purity data for the other thiocarbamates synthesized [vernolate (S-propyl-N,Ndipropylthiocarbamate) (1b), ethiolate (S-ethyl-N,N-diethylthio-

Zimmerman et al. carbamate) (1c), EPTC (S-ethyl-N,N-dipropylthiocarbamate) (1d), butylate (S-ethyl-N,N-diisobutylthiocarbamate) (1e), and S-methyl-1-piperidinecarbothioate) (1f)] are available in the Supporting Information. Synthesis of Thiocarbamate Sulfones. The general procedure used for the synthesis of the thiocarbamate sulfones is described using for molinate sulfone (S-ethyl-hexahydro-1Hazepine-1-carbothioate sulfone) (4a). A solution of molinate (0.94 g, 5 mmol) in dichloromethane (10 mL) was cooled in an ice bath, and m-chloroperoxybenzoic acid (2.70 g, 11 mmol) in the same solvent (25 mL) was added with stirring. The reaction was stirred at 0 °C for 4 h, the solid was filtered, and the filtrate was washed with saturated NaHCO3 (2 × 25 mL) and then dried and concentrated using a rotary evaporator. The product was purified using column chromatography (hexane; 9:1 hexane/ ethyl acetate; 5:1 hexane/ethyl acetate), and fractions containing the product were combined, and the solvent was removed under reduced pressure yielding a colorless liquid. 1H NMR (CDCl3): δ 1.36 (t, 3H, CH3), 1.57 (m, 4H, ring CH2), 1.78 (m, 4H, ring CH2), 3.30 (q, 2H, CH2CH3), 3.49 (t, 2H, CH2-N), 3.84 (t, 2H, CH2-N). 13C NMR (CDCl3): δ 6.7 (CH3), 26.0, 26.3, 27.3, 28.8 (ring CH2), 46.1 (CH2CH3), 46.8 and 48.8 (CH2-N), 160.6 (Cd O). Spectral and purity data for the other thiocarbamate sulfones synthesized [S-propyl-N,N-dipropyl thiocarbamate sulfone (4b), S-ethyl-N,N-diethylthiocarbamate sulfone (4c), Sethyl-N,N-dipropylthiocarbamate sulfone (4d), S-ethyl-N,Ndiisobutylthiocarbamate sulfone (4e), S-methyl-1-piperidinecarbothioate sulfone (4f)] are available in the Supporting Information. Synthesis of Thiocarbamate Cysteine Conjugates (10). All thiocarbamate cysteine conjugates were prepared according to a previously published procedure as described here for HHACCys (10a). Briefly, the pH of a solution of L-cysteine (0.52 g, 4 mmol) in water was adjusted to 8 with 1 N NaOH and stirred with molinate sulfone (0.44 g, 2 mmol) in methanol for 24 h. After the methanol was removed, purification was achieved by HPLC by injecting the reaction mixture onto a C18 PRP-1 column (Hamilton, 3.9 mm × 300 mm, 300 Å). The desired product was eluted at a flow rate of 2.0 mL/min using a linear gradient of 10-90% B over 10 min and then maintained at 90% B for 9 min before returning to initial conditions. Solvent A contained 5 mM formic acid in water, and solvent B contained 5 mM formic acid in ACN. The separation was monitored at 214 nm, and fractions containing the product were combined and lyophilized leaving a white solid. 1H NMR (CDCl3): δ 1.48 (m, 4H, CH2), 1.65 (m, 4H, CH2), 3.26 (q, 2H, CH2), 3.64 (m, 4H, CH2), 3.96 (q, 1H, CH). 13C NMR (CDCl3): δ 26.4, 26.8, 27.36 and 27.07 (ring CH2), 30.7 (S-CH2), 48.4 and 48.7 (ring CH2), 55.2 (CH-NH2), 169.2 (CdO), 172.6 (N-CdO). ESI-MS m/z 247 (M + H)+. Spectral and purity data for the other thiocarbamate cysteine conjugates synthesized [VER-Cys (10b), ETH-Cys (10c), EPTC-Cys (10d), BUT-Cys (10e), and PIP-Cys (10f)] are available in the Supporting Information. Chromatography. Analysis of intact globin samples was performed on a Waters 2690 HPLC system. Globin (1.8-2.5 mg) from non-exposed and thiocarbamate-exposed rats was dissolved in 0.1% TFA (750 µL). The absorbance was measured at 280 nm and diluted as necessary to produce a final absorbance of 1. Fifty microliters was injected onto a C4 RP column (4.6 mm × 150 mm, 5 µm, 300 Å, Phenomenex, Torrance, CA). Globin samples were eluted at a flow rate of 1 mL/min with a linear gradient from initial conditions of 56% A [solvent A ) ACN/ H2O/TFA (20:80:0.1%)] and 44% B [solvent B ) ACN/H2O/TFA (60:40:0.08%)] to 30% A and 70% B over 30 min while monitoring at 214 nm. Globin R- and β-chains were separated and collected for MALDI-TOF MS and LC/MS/MS using a Shimadzu LC-10ADvp connected to an SPD-10A detector with HP Chemstation software. Globin collected from non-exposed or thiocarbamateexposed rats was dissolved in solvent A to a final concentration of 10 mg/mL, and 50-100 µL was loaded onto a C4 Waters Delta Pak column (3.9 mm × 300 mm, 300 Å). Protein fractions were

Thiocarbamate Adducts and Enzyme Inhibition eluted at a flow rate of 2 mL/min with a linear gradient from initial conditions of 56% A [solvent A ) ACN/H2O/TFA (20:80: 0.1%)] and 44% B [solvent B ) ACN/H2O/TFA (60:40:0.08%)] to 40% A and 60% B over 30 min while monitoring at 214 nm. Protein fractions were collected, and ACN was removed and then lyophilized before analysis with MALDI-TOF MS. Determination of Esterase Activity. Esterase activity was measured in plasma, brain mitochondria (BMT), and both liver and testes mitochondrial and microsomal fractions in control and thiocarbamate-treated rats by monitoring the hydrolysis of p-nitrophenylacetate spectrophotometrically at 400 nm using a procedure modified for a microplate reader (4, 20). The assay mixture (final volume 225 µL) contained 0.2 mg of protein in buffer (0.1 M potassium phosphate buffer, pH 7.4, 1 mM EDTA, and 0.5 mM DTT), and the reaction was initiated by the addition of the p-nitrophenylacetate in ACN (10 µL of 22.5 mM, 1 mM final concentration). Protein content was determined by the method of Bradford using BioRad reagent dye and bovine serum albumin as the standard. ALDH Activity. The ALDH activity in control and thiocarbamate-treated rats was measured by following the formation of NADH fluorimetrically for brain with excitation at 340 nm and emission at 450 nm and spectrophotometrically for the liver and testes at 340 nm using procedures modified for a microplate reader (21, 22). The assay mixtures contained 50 mM sodium pyrophosphate (pH 8.8), pyrazole (80 µM final concentration added in 10 µL of pyrophosphate buffer), rotenone (2 µM final concentration added in 1.4 µL of methanol), sodium deoxycholate (0.25 mg/mg of protein in pyrophosphate buffer), and 0.35-0.5 mg of mitochondrial or microsomal protein. Buffer was added to produce a final volume of 700 µL. Triplicate aliquots (175 µL) of the mixture were transferred into microplate wells. NAD+ (0.5 mM final concentration added in 25 µL of pyrophosphate buffer) was added, the reaction was initiated by the addition of the substrate propionaldehyde (50 µM and 5 mM final concentrations in 25 µL of pyrophosphate buffer), and the formation of NADH was monitored for 3-5 min. Acid Hydrolysis. Samples for LC/MS/MS analysis were prepared using mitochondria and microsomes from brain, liver, and testes from control and treated rats. Protein (1-10 mg) was lyophilized and hydrolyzed in a chamber containing 200 µL of 6 N HCl, flushed with argon gas, and hydrolyzed for 18 h at 110 °C. Following completion of hydrolysis, samples were reconstituted in 100 µL of ACN/H2O/formic acid (10:90:0.02%) and filtered using a Costar Spin-X centrifuge spin filter (0.2 µm pore size). Samples were stored at -20 °C until analysis. Protein content was determined by the method of Bradford with BioRad reagent dye using bovine serum albumin as a standard. Mass Spectrometry. MALDI-TOF MS analysis of intact proteins was performed on a Voyager Elite (PerSeptive Biosystems, Inc.) time-of-flight mass spectrometer. The ion acceleration was 25 kV, the grid voltage was 95% for intact proteins, and the guide wire voltage was 0.1%. Mass spectra were acquired as the sum of ions generated by irradiation of the sample with 100-300 laser pulses and processed using GRAMS/ 386 software (Galactica Industries Corp., Salem, NH). A saturated solution of SA in ACN/H2O/TFA (50:49:0.1 v/v/v) was used as the matrix. For analysis of intact proteins, 2 µL of a globin mixture (1 mg/mL) was added to 2 µL of the SA matrix and applied to the plate by the dried droplet technique and air-dried. For analysis of the purified protein fractions, 2-4 µL of the mixture was added to an equivalent volume of the SA matrix and applied to the sample plate in a similar manner. Myoglobin was used as an internal standard with an m/z of 16 952.5. LC/MS/MS analysis of the R-Cys adduct with SRM was performed using a Waters 2690 HPLC system coupled with a Finnigan TSQ 7000 triple-quadrupole ESI/MS (Finnigan, San Jose, CA). A PE Biosystems (Foster City, CA) 785A Programmable Absorbance UV detector was used and set to monitor at 214 nm. The electrospray voltage was maintained at 4 kV, and the capillary temperature was held at 200 °C. CID occurred in Q2 at a collision energy of 20 eV with the collision gas (argon)

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 261 at 2.0 mT. Solvent A contained 5 mM formic acid in water, and solvent B contained 5 mM formic acid in ACN for molinate-, vernolate-, EPTC-, and butylate-treated samples or 5 mM formic acid in methanol for ethiolate-treated samples. LC separations were performed on a C18 PRP-1 column (2.1 mm × 100 mm, 300 Å, Hamilton, Reno, NV). Separations for molinate, vernolate, EPTC, and butylate samples were achieved using initial conditions of 10% B (90% A) held for 10 min followed by a linear gradient to 90% B (10% A) over 5 min and held for 7 min before returning to initial conditions. Equilibration with 10% B (90% A) was allowed for 15 min, and the total run time was 38 min. Separations for ethiolate samples were achieved using initial conditions of 10% B (90% A) followed by a linear gradient to 90% B (10% A) over 7 min and held for 10 min before returning to initial conditions. Equilibration with 10% B (90% A) was allowed for 15 min, and the total run time was 33 min. Twentyfive microliters of the sample treated with 5 µL of the internal standard (80 ng/µL) was injected, and the eluant was directed into the mass spectrometer using a switching valve. SRM experiments, used to detect cysteine adducts, monitored the following transitions: m/z 247f126 for HHAC-Cys; m/z 249f128 for both VER-Cys and EPTC-Cys; m/z 277f156 BUT-Cys; m/z 221f110 for ETH-Cys; and m/z 233f112 for PIP-Cys, the internal standard. The scan width for the daughter ions was 1 amu, and the total cycle time was 2 s. The ratios of the resulting peak areas were used to calculate the amount of each N,Ndialkylaminocarbonyl-cysteine adduct in the acid hydrolysates by reference to a calibration curve. A calibration curve was generated by dissolving varying amounts of the authentic N,Ndialkylaminocarbonyl-cysteine in solvent A and adding a fixed amount of PIP-Cys so each 25 µL injection would contain 0-1000 ng of N,N-dialkylaminocarbonyl-cysteine and 100 ng of PIP-Cys. The ratio of the area of analyte to the internal standard was plotted against the concentration of N,N-dialkylaminocarbonyl-cysteine in the sample to generate a standard curve for each thiocarbamate. Molinate Neurotoxicity Assessment. Rats were exposed via 2ML2-wk Alzet osmotic pumps surgically implanted in the abdomen or subcutaneously between the scapulae while under deep anesthesia (90 mg/kg ketamine HCl and 7.5 mg/kg xylazine). The pumps were surgically replaced after 2 weeks to achieve longer exposures. Molinate was delivered as a mixture of molinate in PEG 200 adjusted to obtain the correct dose. Control animals received PEG 200 alone. All solutions were filtered through a sterile 0.22 µm syringe filter prior to filling the pumps. Rats administered molinate via abdominal pumps (three treated 230-242 g bwt and three controls 355-392 g bwt) received 1 mmol/kg/day for 46 days. Rats administered molinate using subcutaneous osmotic pumps (two treated 374, 438 g bwt and one control 462 g bwt) received 0.5 mmol/kg/day for 59 days. Before, during, and at the end of the exposure periods, blood samples were obtained from a tail vein and the amount of modified β-globin was determined using HPLC as described above. At the end of the exposure periods, the animals were perfused through the left ventricle of the heart under deep anesthesia (100 mg/kg ketamine HCl and 15 mg/kg xylazine) with a solution of 0.9% NaCl, 0.2% sodium nitroprusside, and 2.5% poly(vinylpyrrolidone) followed by 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Sections of sciatic nerve, posterior tibial nerve, and the muscular branch of the posterior tibial nerve from both hind legs were excised and immersion fixed in 4% glutaraldehyde for 1 h and then transferred to cacodylate buffer, pH 7.2. Embedding and staining was performed as described (23). Thick (1 µm) sections of peripheral nerve tissue were evaluated by light microscopy on an Olympus BX41 microscope.

Results Intact Globin Analyis by MALDI-TOF MS and RPHPLC. HPLC chromatograms of globin samples obtained

262

Chem. Res. Toxicol., Vol. 17, No. 2, 2004

Figure 1. HPLC chromatograms of rat globin samples separated by reverse phase chromatography on a C4 PRP-1 column. (A) Representative chromatograph of control globin showing a single peak for the two R-chains eluting at 18 min and two peaks for the four β-chains eluting between 22 and 25 min and an undetermined late eluting peak (31.5 min) that was also observed in samples obtained from both control and thiocarbamate-exposed rats. (B) Chromatograph of globin isolated from vernolate-treated rats produced an additional peak to those seen in the control sample that eluted at 28 min.

from control and thiocarbamate-treated rats contained a single peak corresponding to the two native R-chains and two peaks corresponding to the four native β-chains of globin. The globin samples obtained from the thiocarbamate-treated rats each produced an additional peak eluting after the native β-chains that was not observed in the control samples. Representative chromatograms are shown in Figure 1. MALDI-TOF MS analysis of the control globin resolved two R-chains at m/z 15 150 and 15 197 (Figure 2A) and a single peak for the β-chains at m/z 15 849. The additional late eluting peak observed in each of the thiocarbamate globin samples was collected and analyzed by MALDI-TOF MS. MALDI-TOF MS analysis of the late eluting HPLC peaks of the thiocarbamate-treated samples demonstrated the new peaks to have an m/z of 15 975 for molinate, m/z of 15 977 for both vernolate and EPTC, and m/z of 15 949 for ethiolate (Figure 2B-E), consistent with the addition of the corresponding N,N-dialkylaminocarbonyl adduct to the β3-chain of globin. The quantity of the modified β3-globin measured by RP-HPLC expressed as a percentage of the total β-globin chains for each compound is presented in Table 1. In contrast, MALDI-TOF analysis of the late eluting fraction obtained from butylate-treated rats yielded an m/z of 15 849 (Figure 2F), identical to that of the native combined β-chain peaks. Also consistent with the absence of detectable adduct was the finding of no significant increase in the modified β-peak area relative to the controls for butylate (Table 1).

Zimmerman et al.

Enzyme Activities. The effects of molinate, vernolate, ethiolate, EPTC, and butylate on plasma and tissue esterase activities are presented in Figure 3. In plasma, all compounds except EPTC were associated with small but statistically significant increases in esterase activity relative to controls. For other samples, esterase activity in controls was tissue-dependent and exhibited about a 5-fold range among brain, liver, and testes. Thiocarbamate-mediated esterase inhibition was both tissue- and compound-dependent and was observed for all samples other than plasma. Most of the compounds produced from 60 to 80% inhibition but ranged from no significant inhibition for butylate in brain mitochondria (BMT), liver mitochondria (LMT), and liver microsomes (LMC) to almost 90% for ethiolate in BMT. For four out of the five samples, molinate was one of the two compounds producing the greatest amount of inhibition, and butylate produced the least amount of esterase inhibition. Effects of the thiocarbamates on ALDH activity in vivo were examined in brain, liver, and testes samples. Low Km ALDH activity was determined in liver and testes mitochondria (TMT) whereas total ALDH activity was assessed in BMT and liver and testes microsomes (TMC) due to the low levels of low Km activity in control animals. From Figure 4, it can be seen that ALDH activity in controls covered about a 10-fold range across the samples examined. In general, there was less variability in the observed ALDH inhibition among the compounds for a particular tissue as compared to those observed for esterase activity with the exception of LMC. Analysis of S-(N,N-Dialkylaminocarbonyl)cysteine Adducts (10) by LC/MS/MS. Acid hydrolysates of mitochondrial and microsomal preparations from brain, liver, and testes of control and thiocarbamatetreated animals were analyzed for the formation of the S-(N,N-dialkylaminocarbonyl)cysteine adducts using SRM. The retention times of the synthetic S-(N,N-dialkylamino)cysteine standards were 6.5 min for HHAC-Cys (10a), 12 min for VER-Cys (10b) and EPTC-Cys (10d), 17 min for BUT-Cys (10e), and 4 min for the internal standard (10f) when chromatographed on a C18 PRP-1 column using the conditions described. The synthetic standard for the ethiolate cysteine adduct (10c) had a retention time of 8.6 min, and 9.6 min was the retention time of the PIP-Cys (10f) internal standard when chromatographed in a similar manner using the conditions described. Control hydrolysates from each tissue did not contain a peak for any of the S-(N,N-dialkylamino)cysteine adducts being measured, although the internal standard could be observed. Hydrolysates of samples obtained from thiocarbamate-treated rats for each tissue and specific fraction contained peaks having the same retention times as the respective authentic synthetic standard being measured, although the levels of adducts varied significantly for each of the thiocarbamates. The quantity of adducts detected in the brain, liver, and testes is shown in Figure 5. Estimates of the amount of adduct were determined based upon a calibration curve generated from response ratios of known quantities of the authentic standard for each of the thiocarbamates examined to a constant amount of internal standard. The lowest levels of protein adducts were observed in BMT samples. The compound most consistently producing the lowest level of protein adducts was butylate ranging from none detected to 2.3 pmol/mg protein. TMT

Thiocarbamate Adducts and Enzyme Inhibition

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 263

Figure 2. MALDI-TOF MS spectra of globin. (A) Globin isolated from control rats containing peaks for the two R-chains at m/z 15 150 and 15 197 and a single peak for the β-chains at m/z 15 849. Analysis of the late eluting peaks isolated from globin samples obtained from rats administered: (B) molinate (m/z 15 975) consistent with the addition of 126 Da to the β3-chain from the S-hexahydro1H-azepine-1-carbonyl adduct; (C) vernolate (m/z 15 977) consistent with the addition of 128 Da from the S-(N,N-dipropylaminocarbonyl) adduct; (D) ethiolate (m/z 15 949) consistent with addition of 100 Da from the S-(N,N-diethyaminocarbonyl) adduct; (E) EPTC (m/z 15 977) consistent with the addition of 128 Da from the S-(N,N-dipropylaminocarbonyl) adduct; and (F) butylate produced a signal at m/z 15 849 identical to the native combined β-chain peak. Myoglobin was used as an internal standard (m/z 16 952), and SA matrix adducts can be seen as signals 200 or 225 Da larger than the parent protein signals. Table 1. RP-HPLC Measurement of Modified β3-Globina compd

modified β3

compd

modified β3

control molinate vernolate

0.0 (0.0) 11.1 (0.2)* 18.7 (4.4)*

ethiolate EPTC butylate

39.1 (0.8)* 9.1 (0.8)** 1.3 (0.3)

a Modified β -globin expressed as a percent of total β-globin (SE) 3 n ) 4. *p < 0.01 relative to control. **p < 0.05 relative to control.

total β-globin, is shown as a function of exposure duration in Figure 6. The greatest mean level (SE) of modified β3globin, expressed as a percentage of the total β-globin detected in the blood, of the ip molinate-treated rats (1 mmol/kg/day) occurred at the end of the exposure (46 days) and was 15.4% (2.3).

Discussion and LMT had the greatest levels of adducts with molinate and EPTC being associated with the highest levels of modified cysteine in four out of the five tissues examined. EPTC appeared to produce an exceptionally high level of modified cysteine within the LMT relative to other tissues. Molinate Neurotoxicity Assessment. Examination of peripheral nerves from both treatment groups and controls did not reveal any demyelinated axons or myelin edema (micrographs are available in the Supporting Information). Occasional degenerated axons were identified in all groups including controls and were not compound related. The mean level of modified β3-globin that was detected in the blood obtained from the subcutaneous molinate-treated rats, expressed as percent of

All of the thiocarbamate herbicides examined, except for butylate, produced additional peaks in their globin HPLC chromatograms that corresponded to a mass increase consistent with the addition of S-(N,N-dialkylaminocarbonyl)cysteine adducts to the β3-chain of globin as determined by MALDI-TOF mass spectrometry. A considerable difference in the quantity of modified β3globin produced by each of the compounds was observed, ranging from no significant difference relative to controls for butylate up to 39% of the total β-chains for ethiolate suggesting that almost all of β3-chains were modified by ethiolate (Table 1). Of the four compounds examined possessing S-ethyl substitutents, the level of globin modification observed decreased with increasing number of carbons attached to nitrogen with the cyclic six carbon

264

Chem. Res. Toxicol., Vol. 17, No. 2, 2004

Figure 3. Mean nonspecific esterase activities and standard errors in plasma, BMT, LMT, LMC, TMT, and TMC obtained from control (n ) 4) and thiocarbamate-treated (n ) 4/compound) rats. Thiocarbamates in corn oil were administered ip at 100 mg/kg/day for 5 days, and samples were collected 20-24 h after the final dose. Controls were administered corn oil alone. Molinate, vernolate, and ethiolate produced significant (p < 0.01) differences relative to controls for all samples. EPTC produced significant differences (p < 0.01 except p < 0.05 for BMT) in all samples except plasma for which no difference was observed. Butylate produced significant differences in activity only for plasma (p < 0.01), TMC (p < 0.01), and TMT (p < 0.05).

Figure 4. Mean total ALDH activities and standard errors in BMT, LMC, TMC, and mean low Km ALDH activities and standard errors in LMT and TMT. Refer to Figure 3 for treatment, collection, and controls. All compounds produced significant (p < 0.01) differences in activity relative to controls except butylate in LMT (no significant difference) and vernolate in TMC (p < 0.05).

azepine moiety of molinate resulting in lower levels than the six carbon dipropyl substituent on EPTC. Butylate containing the bulkiest diisobutyl groups did not produce any significant levels of modified β3-globin. In contrast, the addition of a methylene to the sulfur substituent resulted in a doubling of the modified β detected, i.e., vernolate vs EPTC (Scheme 1). The relative amounts of adduct measured for globin presumably reflect the relative ability of each compound to undergo oxidative metabolism to form a reactive sulfoxide or sulfone followed by nucleophilic addition by a cysteine on globin. In addition to direct conjugation with protein sulfhydryls, evidence has been presented that conjugation with protein sulfhydryls may also proceed through a less direct route involving initial conjugation of the sulfoxide or sulfone metabolite with glutathione (3), followed by cysteine β-lyase-mediated cleavage, methylation, and

Zimmerman et al.

Figure 5. Mean levels and standard errors of S-(N,N-dialkylaminocarbonyl)cysteine measured using LC/MS/MS with SRM in BMT, LMT, LMC, TMT, and TMC. Refer to Figure 3 for treatment, collection, and controls. Quantities of modified cysteine species are expressed per mg of total protein and were calculated from standard curves generated using the response ratios obtained for the synthetic authentic modified cysteine species and the PIP-Cys internal standard.

Figure 6. Modified β-globin formation as a function of molinate exposure duration. Rats were administered either molinate (0.5 mmol/kg/day) in PEG 200 or PEG 200 alone for 59 days using a subcutaneous osmotic pump. Before, during, and at the end of exposure, blood samples were obtained and the amount of modified β-globin was determined as a percent of total β-globin using RP-HPLC.

reoxidation to an S-methylthiocarbamate sulfoxide (5) or sulfone (24). The molinate adduct for globin was previously determined to be located at Cys-125 on the β3- and β2-globin chains (12), a finding identical to that observed for the ETH-Cys adduct produced by disulfiram and N,Ndiethyldithiocarbamate on rat globin (13). Preferential modification at this site is thought to occur as a result of the relatively high reactivity of this cysteine residue that due to its concentration (approximately 8 mM) can act as an effective scavenger of intracellular electrophiles (25). Because of the stability of the adduct, duration of the biological life of globin, and relative ease of sample procurement, detection of S-(N,N-dialkylaminocarbonyl)cysteine on globin may be useful in biomonitoring or forensic applications for thiocarbamate exposure. However, because human hemoglobin lacks the reactive Cys125 residue present in rats, levels are expected to be much lower on human globin and will probably require a more sensitive method than RP-HPLC such as the LC/ MS/MS method presented here. However, the identification of S-(N,N-dialkylaminocarbonyl)cysteine modifications in the mitochondrial and cytosolic preparations examined here supports thiocarbamate-mediated cys-

Thiocarbamate Adducts and Enzyme Inhibition

teine modifications as potential biomarkers for thiocarbamate exposure. Modification of an active site serine or cysteine residue has been proposed as a mechanism for inhibition of esterase or ALDH activity, respectively, by thiocarbamates and disulfiram (4, 10). These conclusions, though, have been primarily based upon the identification of the protein adduct and the relative potency of the oxidized metabolite relative to parent compound. In the current study, the levels of thiocarbamate-mediated covalent protein modifications and the enzyme activities were determined to examine whether protein modifications could be correlated with enzyme inhibition. In the case of ALDH, the cysteine modification examined here corresponds to the adduct proposed to be responsible for modifying the active site (14). Although the lability of the analogous adduct formed on serine precluded its measurement by the methods used in this study, the S-(N,N-dialkylaminocarbonyl)cysteine adduct might reflect generation of the serine adduct to some extent due to the analogous proposed bioactivation pathway preceding formation of the serine adduct (7). Examining the relative potencies for esterase inhibition reveals considerable variability both for a particular compound across the tissue samples examined and among the five thiocarbamates for a particular sample (Figure 3). An unexpected finding was the elevated levels of esterase activity in plasma. There was also no apparent correlation of the amount of adduct measured for a sample to the level of inhibition observed, i.e., the rank order of protein adduct/mg protein did not correspond to the rank order of esterase inhibition observed for a given tissue sample. Two general associations were apparent though, butylate consistently produced relatively low levels of adducts and esterase inhibition, and molinate consistently produced relatively high levels of adducts and esterase inhibition. The observation that vernolate and EPTC were relatively potent inhibitors of esterase in the testes suggests that they too may be capable of producing testicular toxicity similar to molinate whereas butylate would be predicted to be a less potent testicular toxicant based upon its lower potency to inhibit esterases in the testes. Less variability was observed among the thiocarbamates regarding inhibition of ALDH for a given tissue sample relative to that observed for esterase inhibition (Figure 4). Again, the relative potency of a given thiocarbamate varied considerably across the tissue samples assayed. An initial attempt to correlate protein modifications to ALDH inhibition again failed to demonstrate a correspondence of rank order of protein adduct levels to the rank order of percent enzyme inhibition. It is interesting to note though that in contrast to the results obtained for esterase inhibition, molinate was considerably less effective and butylate more effective at inhibiting ALDH activity. For low Km ALDH, data have been presented suggesting that N-monoalkyl thiocarbamate sulfoxide compounds (7) may favor adduct formation at the active site cysteine whereas the dialkyl species may favor modification of other proteins, e.g., esterases (26, 27). Similarly, the analogous S-(ethylaminocarbonyl)cysteine modification has been identified on low Km ALDH in vivo in rats following disulfiram administration and N-dealkylation has been suggested to be a contributing pathway for inhibition of ALDH by disulfiram (14). Thus, the results obtained for ALDH inhibition reported

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 265

here may not only reflect the oxidative metabolism to sulfoxide and sulfone metabolites but also the extent to which each compound is N-dealkylated. This may partially account for the discrepancy in the potency of butylate to inhibit ALDH activity relative to esterase activity and the apparent lack of correlation of the measured adduct levels to ALDH inhibition, as S-methylN-butylthiocarbamate sulfoxide has been reported to be a very potent inhibitor of low Km ALDH (26, 27). Because previous studies characterizing the neuropathy produced by disulfiram demonstrated the formation of S-(diethylaminocarbonyl)cysteine adducts within the nervous system prior to the onset of structural changes, disruption of proteins involved in the maintenance of myelin through sulfhydryl modification was proposed as a putative neurotoxic mechanism for disulfiram (15). The shared bioactivation pathway of the thiocarbamate herbicides and disulfiram that lead to the formation of S-(diethylaminocarbonyl)cysteine adducts suggest that if enzyme inhibition resulting from this modification is responsible for disulfiram neurotoxicity, then the thiocarbamate herbicides may also possess the potential to produce segmental demyelination in the peripheral nervous system. Additionally, because there is a reduced number of steps required for the generation of sulfoxide and sulfone metabolites from thiocarbamates, they may be more effective at cysteine carbamylation and thus potentially more potent neurotoxicants. Data available on the neurotoxicity of thiocarbamates are limited, but there have been a few reports from safety assessment studies associating molinate with the development of lesions within the peripheral and central nervous systems (28). In the current study, the potential of molinate to produce a peripheral neuropathy from similar levels and duration of exposure to those producing demyelination by disulfiram was assessed. The results demonstrated that although molinate produced comparable levels of cysteine modification to those observed in disulfiram-induced neuropathy and was accompanied by ALDH and esterase inhibition, no differences relative to controls were observed regarding morphology in the peripheral nervous system. These findings together with a report that parenteral administration of N,N-diethyldithiocarbamate but not its S-methyl-N,N-diethyldithiocarbamate metabolite produced identical lesions to disulfiram, despite a 2-fold greater level of adducts produced by S-methyl-N,N-diethyldithiocarbamate, argue against a role for cysteine modification in disulfirammediated neuropathy (29). Alternatively, it appears that neurotoxicity is mediated through the dithiocarbamate metabolite of disulfiram, and because a similar metabolite is not produced through metabolism of thiocarbamates, it seems unlikely that thiocarbamates can produce the segmental demyelination observed for dithiocarbamate compounds.

Conclusions All of the thiocarbamates except butylate covalently modified globin with the quantity of modified β-globin being inversely related to the chain length of the nitrogen substituents. In contrast, a clear relationship between the level of S-(dialkylaminocarbonyl)cysteine adducts in the mitochondrial and microsomal samples to the alkyl substituents of the thiocarbamates examined was not evident. Additionally, no clear relationship between the

266

Chem. Res. Toxicol., Vol. 17, No. 2, 2004

level of globin modification and the enzyme inhibition in the tissue samples examined was observed. Although molinate produced relatively high levels of adducts and esterase inhibition and butylate produced low levels of adducts and esterase inhibition, in general, the levels of enzyme inhibition observed for a tissue sample did not appear to be correlated to the levels of S-(N,N-dialkylaminocarbonyl)cysteine adducts within the cytosolic or mitochondrial compartments of the same tissue sample. Therefore, although it appears that cysteine modifications may be of some utility in exposure assessments, they do not appear to reflect the potential of a thiocarbamate to inhibit esterase or ALDH activity. It should be noted that a weakness of the current comparisons is that the total population of proteins within a tissue sample was analyzed. Isolation and analysis of the purified esterase and dehydrogenase enzymes may provide a more valid comparison to evaluate the role of cysteine modification in thiocarbamate-mediated enzyme inhibition. Additionally, it should be noted that the adduct measured in the current study is not that anticipated to be responsible for inhibition of the esterases and does not reflect contributions from the N-dealkylated metabolites. The relatively greater amounts of ALDH inhibition observed for butylate as compared to esterase inhibition suggest that N-dealkylation may make a contribution to ALDH inhibition. From the current data, it appears that molinate, and possibly other thiocarbamates, do not share the neurotoxic potential of dithiocarbamates as a result of the common ability to generate S-(N,N-dialkylaminocarbonyl)cysteine adducts.

Acknowledgment. Chemical synthesis and analyses were performed with the technical guidance of Dr. Venkataraman and Dr. Kalyani Amarnath. LC/MS/MS analysis was performed with the assistance of Lisa Manier of the Mass Spectrometry Research Center at Vanderbilt University. Experiments were performed in part through the use of the VUMC Research EM Resource (sponsored by NIH Grants DK20539 and DK58404). This work was supported by NIEHS Grant ES06387 and by the Center of Molecular Toxicology Grant P30 ES00267. L.J.Z. was supported by NRSA Grant F32 AA05569. Supporting Information Available: Spectral and purity data for synthetic thiocarbamates, thiocarbamate sulfones, and S-(N,N-dialkylaminocarbonyl)cysteine conjugates, micrographs of sciatic nerve sections obtained from molinate-treated, control, and N,N-diethyldithiocarbamate-treated rats, tabular listing of enzyme activities and S-(N,N-dialkylaminocarbony)cysteine adduct levels in thiocarbamate-treated and control rats. This material is available free of charge via the Internet at http:// pubs.acs.org.

References (1) Cashman, J. R., and Olsen, L. D. (1989) S-Oxygenation of Eptam in Hepatic Microsomes from Fresh- and Saltwater Striped Bass (Morone saxatillis). Chem. Res. Toxicol. 2, 392-399. (2) Cashman, J. R., and Olsen, L. D. (1990) S-Oxygenation of Thiobencarb (Bolero) in Hepatic Preparations from Striped Bass (Morone saxatilis) and Mammalian Systems. Chem. Res. Toxicol. 3, 433-440. (3) Tjeerdema, R. S., and Crosby, D. G. (1988) Disposition, biotransformation, and detoxification of molinate (Ordram) in whole blood of the common carp (Cyprinus carpio). Pestic. Biochem. Physiol. 31, 24-35. (4) Ellis, M. K., Richardson, A. G., Foster, J. R., Smith, F. M., Widdowson, P. S., Farnworth, M. J., Moore, R. B., Pitts, M. R., and Wickramaratne, G. A. de S. (1998) The reproductive toxicity

Zimmerman et al.

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

of molinate and metabolites to the male rat: Effects of testosterone and sperm morphology. Toxicol. Appl. Pharmacol. 151, 2232. Sancho, E., Ceron, J. J., and Ferrando, M. D. (2000) Cholinesterase activity and hematological parameters as biomarkers of sublethal molinate exposure in Anguilla anguilla. Ecotoxicol. Environ. Saf. 46, 81-86. Jewell, W. T., Hess, R. A., and Miller, M. G. (1998) Testicular toxicity of molinate in the rat: Metabolic activation via sulfoxidation. Toxicol. Appl. Pharmacol. 149, 159-166. Jewell, W. T., and Miller, M. G. (1998) Identification of a carboxylesterase as the major protein bound by molinate. Toxicol. Appl. Pharmacol. 149, 226-234. Casida, J. E., Kimmel, E. C., Ohkawa, H., and Ohkawa, R. (1975) Sulfoxidation of Thiocarbamate Herbicides and Metabolism of Thiocarbamate Sulfoxides in Living Mice and Liver Enzyme Systems. Pestic. Biochem. Physiol. 5, 1-11. Zemaitis, M. A., and Greene, F. E. (1976) Impairment of Hepatic Microsomal and Plasma Esterases of the Rat by Disulfiram and Diethyldithiocarbamate. Biochem. Pharmacol. 25, 453-459. Hart, B. W., and Faiman, M. D. (1995) Inhibition of rat liver low Km aldehyde dehydrogenase by thiocarbamate herbicides. Occupational implications. Biochem. Pharmacol. 49, 157-163. Quistad, G. B., Sparks, S. E., and Casida, J. E. (1994) Aldehyde dehydrogenase of mice inhibited by thiocarbamate herbicides. Life Sci. 55, 1537-1544. Zimmerman, L. J., Valentine, H. S., Amarnath, K., and Valentine, W. M. (2002) Identification of a S-Hexahydro-1H-azepine-1carbonyl adduct produced by molinate on Rat Hemoglobin β2 and β3 Chains in vivo. Chem. Res. Toxicol. 15, 209-217. Erve, J. C., Jensen, O. N., Valentine, H. S., Amarnath, V., and Valentine, W. M. (2000) Disulfiram generates a stable N,Ndiethylcarbamoyl adduct on Cys-125 of rat hemoglobin. Chem. Res. Toxicol. 13, 237-244. Shen, M. L., Johnson, K. L., Mays, D. C., Lipsky, J. J., and Naylor, S. (2001) Determination of in vivo adducts of disulfiram with mitochondrial aldehyde dehydrogenase. Biochem. Pharmacol. 61, 537-545. Tonkin, E. G., Erve, J. C. L., and Valentine, W. M. (2000) Disulfiram produces a non-carbon disulfide-dependent Schwannopathy in the rat. J. Neuropathol. Exp. Neurol. 59, 786-797. Petterson, H., and Tottmar, O. (1982) Inhibition of Aldehyde Dehydrogenase in Rat Brain and Liver by Disulfiram and Coprine. J. Neurochem. 39, 628-634. Lake, B. G. (1987) Preparation and characterization of microsomal fractions for studies on xenobiotic metabolism. In Biochemical Toxicology, a Practical Approach (Snell, K., and Mullock, B., Eds.) pp 183-215, IRL Press, Oxford. Tottmar, S., Petterson, H., and Kiessling, K. H. (1973) The subcellular distribution and properties of aldehyde dehydrogenase in rat liver. Biochem. J. 135, 577-586. Feingold, I. B., Longhurst, P. A., and Colby, H. D. (1993) Regulation of adrenal and hepatic R-tocopherol content by androgens and estrogens. Biochim. Biophys. Acta 1176, 192-196. Morgan, E. W., Yan, B., Greenway, D., and Petersen, D. R. (1994) Purification and characterisation of two rat liver microsomal carboxylesterases (Hydrolase A and B). Arch. Biochem. Biophys. 315, 495-512. Nelson, A. N., and Lipsky, J. J. (1995) Microtiter Plate-based Determination of Multiple Concentration-Inhibition Relationships. Anal. Biochem. 231, 437-439. Mays, D. C., Nelson, A. N., Fauq, A. H., Shriver, Z. H., Veverka, K. A., Naylor, S., and Lipsky, J. J. (1995) S-methyl N,Ndiethylthiocarbamate sulfone, a potential metabolite of disulfiram and potent inhibitor or low Km mitochondrial aldehyde dehydrogenase. Biochem. Pharmacol. 49, 693-700. Johnson, D. J., Graham, D. G., Amarnath, V., Amarnath, K., and Valentine, W. M. (1998) Release of carbon disulfide is a contributing mechanism in the axonopathy produced by N,N-diethyldithiocarbamate. Toxicol. Appl. Pharmacol. 148, 288-296. Staub, R. E., Sparks, S. E., Quistad, G. B., and Casida, J. E. (1995) S-Methylation as a bioactivation mechanism for mono- and dithiocarbamate pesticides as aldehyde dehydrogenase. Chem. Res. Toxicol. 8, 1063-1069. Rossi, R., Barra, D., Bellelli, A., Boumis, G., Canofeni, S., Simplicio, P. D., Lusini, L., Pascarella, S., and Amiconi, G. (1998) Fast-reacting thiols in rat hemoglobins can intercept damaging species in erythrocytes more efficiently than glutathione. J. Biol. Chem. 273, 19198-19206. Staub, R. E., Quistad, G. B., and Casida, J. E. (1998) Mechanism for Benomyl Action as a Mitochondrial Aldehyde Dehydrogenase Inhibitor in Mice. Chem. Res. Toxicol. 11, 535-543.

Thiocarbamate Adducts and Enzyme Inhibition (27) Staub, R. E., Quistad, G. B., and Casida, J. E. (1999) S-Methyl N-Butylthiocarbamate selective carbamoylating agent for mouse mitochondrial aldehyde dehydrogenase Sulfoxide. Biochem. Pharmacol. 58, 1467-1473. (28) Cochran, R. C., Formoli, T. A., Pfeifer, K. F., and Aldous, C. N. (1997) Characterization of risks associated with the use of molinate. Regul. Toxicol. Pharmacol. 25, 146-157.

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 267 (29) Tonkin, E. G., Valentine, H. L., Zimmerman, L. J., and Valentine, W. M. (2003) Parenteral N,N-diethyldithiocarbamate produces segmental demyelination in the rat that is not dependent on cysteine carbamylation. Toxicol. Appl. Pharmacol. 189, 139-150.

TX034209C