254
Chem. Res. Toxicol. 1995,8, 254-261
Characterization of Protein Adducts Produced by N-Methyldithiocarbamateand N-Methyldithiocarbamate Esters William M. Valentine,* Venkataraman Amamath, Kalyani Amarnath, and Doyle G. Graham Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 Received September 27, 1994@
The toxicity of N-methyldithiocarbamate may be mediated through decomposition to more biologically active compounds. Two principal products, CS:! and methyl isothiocyanate, have the potential to interact covalently with macromolecules in biological systems. In this investigation the ability of N-methyldithiocarbamate to generate methyl isothiocyanate and CS:! under physiological conditions resulting in acylation and covalent cross-linking of proteins was examined using 13C NMR and GC/MS. Two N-methyldithiocarbamate esters, 5'-methyl N-methyldithiocarbamate and (N-acetyl-S-methylthiocarbamoyl)cysteine, were also investigated to evaluate the acylating ability of sulfhydryl conjugates of N-methyldithiocarbamate. The predominant and most stable adduct produced by the free dithiocarbamate and its S-substituted esters was methylthiourea on E-lysyl and N-terminal a-amino groups. Derivatization on N-terminal amino groups progressed more rapidly for the dithiocarbamate than for its mercapturate. Methylurea protein adducts were also produced by the dithiocarbamate and its esters, suggesting production of methyl isocyanate in the decomposition of N-methyldithiocarbamate. Covalent cross-linking of ,&lactoglobulin by N-methyldithiocarbamate resulting from its decomposition to CS:! was observed using denaturing polyacrylamide gel electrophoresis. These results demonstrate the ability of a monoalkyldithiocarbamate to acylate protein amino groups and effect covalent cross-linking. These processes represent molecular mechanisms that may contribute to the toxicity of this class of compounds.
Introduction Dithiocarbamates play a prominent role in pest control and industrial applications (11. Their use on food crops and as medicinal agents results in the exposure of a considerable portion of the world's population. In addition, both animal and plant wildlife may be exposed through pesticidal use or accidental release into the environment (2). Teratogenic (31, genotoxic (4, 5), and neurotoxic (6) effects have been associated with exposure to dithiocarbamates, and the general cytotoxicity of one monoalkyldithiocarbamate, N-methyldithiocarbamate (NMDC),' is exemplified by its use as a soil fumigant (7). The toxicity of NMDC to higher forms of life is also evident from the large number of sporting fish and other aquatic life killed following the accidental introduction of a commercial product (metam sodium) containing NMDC into the Sacramento River (8). The compounds produced through decomposition of NMDC have been studied experimentally (9) and in the environment (21, but the interactions of these decomposition products with macromolecules have not been fully determined. A more complete description of the interactions of dithiocarbamates and their decomposition products with macromolecules in biological systems will aid in the delineation of the molecular mechanisms of toxicity for dithiocarbamates.
* Address correspondence to this author. Phone: 919-684-3217; Fax: 919-684-3324; Express Mail: MSRB Room 199,Duke University Medical Center, Durham, NC 27710. @Abstractpublished in Advance ACS Abstracts, January 15, 1995. Abbreviations: NMDC, N-methyldithiocarbamate;SDS, sodium dodecyl sulfate; MTBSTFA, N-methyl-N-(tert-butyldimethylsily1)trifluoroacetamide; TBDMCS, tert-butyldimethylchorosilane;TBDMS, tert-butyldimethylsilyl group.
Previous investigations have suggested that two breakdown products, CS2 and isothiocyanate, may be responsible for the toxic effects exerted by monoalkyldithiocarbamates (9). Isothiocyanates have received considerable attention due to their natural occurrence as glucosinolates in cruciferous vegetables and their potential anticarcinogenic properties (I0, 11). The electrophilic character of isothiocyanates makes them susceptible to nucleophilic addition by sulfhydryl and amino moieties of proteins, yielding dithiocarbamate ester or thiourea, respectively (12). Such chemical modifications of lysyl or cysteinyl residues of proteins could interfere with electrostatic and hydrogen bonding interactions of proteins vital to their functions. Dithiocarbamates may also serve as substantial sources of CS2 with the ability to release CS2 gradually over extended periods of time. The toxicity of CS2 is well established (13, 14) although its underlying mechanisms of action remain to be determined. One potential mechanism through which CS2 may exert toxicity is covalent cross-linking of proteins subsequent to dithiocarbamate formation on protein amino groups (15,16). In this study the ability of a commonly used monoalkyldithiocarbamate, NMDC, to decompose to isothiocyanate and CS2 under physiological conditions resulting in formation of protein adducts and covalent cross-linking was examined. The structures of the decomposition products and adducts formed on model proteins were characterized using 13C NMR and GCNS. Because conjugation of dithiocarbamates and isothiocyanates with sulfhydryl functions is expected to be both reversible under physiological conditions and an important route of biotransformation (7,17-19), the decomposition prod-
Q893-228x/95/27Q8-Q254$09.0QlQ0 1995 American Chemical Society
Methyldithiocarbamate Protein Adducts ucts and adducts formed by S-linked dithiocarbamate esters of NMDC were also examined. Release of CS2 by NMDC resulting in protein cross-linking was assessed using denaturing polyacrylamide gel electrophoresis.
Experimental Procedures General. NMR measurements were performed with either a General Electric GN-300 WB or a Varian Unity 500 spectrometer. The parameters for protein spectra obtained on the GN-300 WB were described previously (25). Nominal parameters for 13CNMR spectra acquired a t 125 MHz without proton decoupling in the double precision mode were as follows: sweep width 29 000 Hz, pulse repetition 1.8 s, 10 000 acquisitions, 10 Hz line broadening, and 131 072 data points. Spectra obtained on the GN-300 WB and Varian Unity used 20 and 10 mm sample tubes, respectively. Deuterium oxide was used as an internal lock signal. All chemical shifts were referenced to external aqueous 3-(trimethylsilyl)-l-propanesulfonicacid and adjusted t o TMS scale by subtraction of 1.7 ppm with an experimental error of &lppm for protein samples and f 0 . 1 ppm for all other solutions. All GCNS analyses, except where specified otherwise, were performed on a Hewlett-Packard 5890 Series I1 gas chromatograph connected to a 5971A mass selective detector using electron impact ionization. The column (HP-5; 25 m x 0.33 mm x 0.25 pm) was used with temperature programming. A JEOL JMS SX-102 high resolution mass spectrometer was used for FAB/MS with 3-mercapto-1,2-propanediolas a matrix and polyethylene glycol 600 as an internal standard. Chemicals. Caution: Carbon disulfide is volatile, f l a m mable, irritating to the skin, and toxic; gloves and a f u m e hood should be used when handling this compound. [13C]Carbon disulfide (99%) was acquired from Cambridge Isotope Laboratories (Cambridge, MA). Bovine serum albumin fraction V (9699%) and P-lactoglobulin were obtained from Sigma Chemical Co. (St. Louis, MO). All the other chemicals were from Janssen Chimica (New Brunswick, NJ). Sodium NMDC (6). Methylamine (1.75 mL of a 12 M solution, 20 mmol) was taken in water (20 mL) and ethanol (20 mL), and an electrode was immersed for monitoring the pH. Carbon disulfide (1.8 mL, 30 mmol) was added, and the reaction solution was stirred at room temperature. The pH of the solution was maintained in the range 9.5-10 with 4 N NaOH. When the pH did not change, the reaction solution was concentrated to 5 mL and the rest of the moisture was allowed to evaporate slowly. The solid was collected and checked by its W spectrum (maxima a t 282 and 251 nm). S-MethylN-Methyldithiocarbamate(7a). At the end of the reaction from the previous step, the solution was concentrated to 20 mL and cooled in ice. Iodomethane (1.9 mL, 30 mmol) was added along with methanol (20 mL), and the reaction mixture was stirred in ice for 1 h and at room temperature for another h. The reaction solution was concentrated, and the residue was purified by column chromatography (3:l hexaneethyl acetate). The fractions containing the ester were combined and evaporated to a clear viscous liquid: 2 g (83%). (N-Acetyl-S-methylthiocarbamoy1)cysteine(7b). NAcetylcysteine (1.68 g, 10 mmol) was dissolved in water (40 mL) and the pH raised to 9 with 1N NaOH. Methyl isothiocyanate (0.8 g, 11mmol) was added, and the solution was stirred at room temperature for 2 h. The reaction mixture was washed with ethyl acetate (3 x 15 mL), and the product was purified by flash chromatography (20% water in acetonitrile): 1,- ( E ) a t 270 nm 8080; 13C NMR 6 23.3 (CHsCO), 35.2 (CHsN), 37.8 (C-3), 55.6 (C-2), 174.6 (CHsCO), 177.3 (COzH), 198.6 (C=S). 13C-Labeling of Compounds. Methylamine (50 pL, 0.6 mmol) was taken in a water-ethanol mixture (2 mL each) and cooled in ice. [13ClCarbondisulfide (30 pL, 0.5 mmol) was added with stirring, and the stirring was continued for 5 h while 0.5 N NaOH (1 mL) was added in small portions. The pH of the solution was lowered to 7.2 with NaHzPO4. Ethanol and most of the water were removed in vacuo. The residue was dissolved
Chem. Res. Toxicol., Vol. 8, No. 2, 1995 255 in 0.1 M phosphate buffer (pH 7.51, and the concentration was estimated spectrophotometrically. For preparing the 5'-methyl N-methyldithiocarbamate, the reaction mixture from the previous reaction was concentrated to -1 mL and treated with iodomethane (50 pL) in methanol (1 mL). The labeled ester was purified by flash chromatography (3:l hexane-ethyl acetate). Leucine Methylthiohydantoin (3b) and Leucine Methylhydantoin (3a). 3-Methyl-5-(2-methylpropyl)-2-thioimidazolidin-4-one (methylthiohydantoin of L-leucine) was prepared as described (20);mp 149-151 "C. For preparing the hydantoin 3a, methyl isocyanate (240 pL, 4 mmol) was added to a solution of L-leucine (524 mg, 4 mmol) in water (20 mL, pH adjusted to 8 with 1 N NaOH) and stirred at room temperature for 2 h. Acetic acid (7 mL) and concentrated hydrochloric acid (13 mL) were added, and the mixture was heated at 100 "C for 1h. The solution was concentrated, and the white solid was crystallized from water: 500 mg (66%); mp 129-130 "C. W-(Methylthiocarbamoy1)lysine(4). Na-Acetyllysine (1 g, 5 mmol) was suspended in 2-propanol (20 mL), and enough water was added to get a clear solution. Sodium hydroxide (5 mL of 1N solution) was added followed by methyl isothiocyanate (0.5 g). The solution was stirred a t room temperature for 16 h, washed with ethyl acetate, and evaporated. The residue was purified on a column of silica using water (20%)in acetonitrile. The appropriate fractions (monitored in the same solvent system) were combined and evaporated: 0.8 g; 13CNMR 6 23.22 (CH,CO), 23.68 (C-4), 29.23 (C-5), 31.71 (CH~NHCWS), 32.42 (C-3),45.14 (C-6), 55.81 (C-21, 174.50 (CH3CO),179.70 (COzH), 180.72 (C=S); FAI3/MS m l z 276 (M l)+. The protected thiourea (0.6 g) was hydrolyzed by heating at 110 "C in 6 N HCl (1 mL) for 16 h. The solution was evaporated, and the residue was coevaporated with ethanol. The precipitated solid was collected by centrifugation, washed with ethanol, and dried: 0.4 g; 13C NMR 6 22.79 ((2-41, 29.03 (C-5), 30.77 (C-31, 31.76 (CH3NHC=S), 44.94 (C-6), 54.51 (C-2), 173.23 (COzH), 180.91(C=S); analyzed by MS as TBDMS derivative of 6-isothiocyanatonorleucine (5); CI m l z 417 (M 1); E1 m l z 401 (M CH3), 359 (M - CH3 - tert-butyl), and 257 (M - C02-TBDMS). Decomposition of ti3C=S1NMDC. A 12 mL solution containing 0.1 M sodium phosphate (pH 7.5), DzO (10% vlv), and [13C=S]NMDC (0.25 mmol) was incubated at 37 "C and monitored by 13C NMR spectroscopy. At the end of the incubation the solution was extracted with ethyl acetate and analyzed by GC/MS. Adduct Formation on Bovine SerumAlbumin by [W=S]"MDCand S-MethylN-Methyl[W=S]dithiocarbamate.A 12 mL solution containing bovine serum albumin (4% w/v), D20 (10% vlv), 0.1 M sodium phosphate (pH 7.51, and either [W=S]NMDC (0.25 mmol) or I3C-labeled7a (0.25 mmol) was incubated at 37 "C and monitored by 13CNMR spectroscopy. At the end of incubation the protein solutions were dialyzed against 0.1 M sodium phosphate (pH 7.4) using dialysis tubing (molecular wt cutoff 3500) and then concentrated in vacuo. Portions of the protein solution were then analyzed for the presence of Ne(methyl[13C=S]thiocarbamoyl)lysineby GC/MS subsequent to complete acid hydrolysis (see Figure 1). Estimating N-Terminal Amino Derivatization. ,&Lactoglobulin (5 mg) was dissolved in 88% formic acid (2 mL) a t room temperature for 2 days. Formic acid was removed in vacuo, and the residue was dissolved in water (2 mL) and extracted with ethyl acetate (3 x 2 mL). The combined extracts were dried, evaporated, and redissolved in 100 pL of ethyl acetate for GC/MS analysis (see Figure 1). A standard curve for the detector response was obtained from solutions containing known concentrations of leucine methylthiohydantoin. Detection of iW(Methylthiocarbamoy1)lysinein the Treated Protein. The protein samples (5-10 mg) were dissolved in 6 N HCl(400 pL) containing a trace of phenol and heated at 110 "C for 20 h. The acid was removed, and the residue was derivatized with MTBSTFA containing 1%TBDMCS in acetonitrile at 100 "C for 1h for GC/MS. Analyses were
+
+
Valentine et al.
266 Chem. Res. Toxicol., Vol. 8, No. 2, 1995
o 1
0;
R = (CH3)zCHCHz
x=o,s X
II
YHCNHCH,
R-~HCNH--.C-CH-NH--. I1 I1 I
NHCNHCH,
It
S
210
Figure 1. Genesis and detection of methylthiourea and methylurea adducts on ,5lactoglobulin and bovine seum albumin. Addition of methyl isothiocyanate or methyl isocyanate to leucine terminal amino groups and lysyl +amino groups (1) generated methylthiourea and methylurea adducts (2). Mild acid hydrolysis (A) of B-lactoglobulin produced leucine methylthiohydantoin and leucine methylhydantoin (3),which were detected by GCMS. Complete protein hydrolysis (B)of bovine serum albumin generated Ne-(methy1thiocarbamoyl)lysine (41, which underwent thermal decomposition to "Ne-lysyl"isothiocyanate (5) following derivatization with TBDMS and analysis by GC/ MS. performed by monitoring multiple ions a t m l z 359, 331, and 257. Rate of Derivatization of p-Lactoglobulin by 6 or 7b. A solution (16 mL) of ,&ladoglobulin (1%w/v in 0.1 M phosphate buffer, pH 7.5) was mixed with NMDC (1.4 mmol) or (N-acetylS-methylthiocarbamoy1)cysteine(1.4 mmol) and incubated a t 37 "C. Aliquots (2 mL) were withdrawn a t various time points, dialyzed against water, lyophilized, and analyzed for derivatization of N-terminal groups by thiohydantoin or hydantoin formation and GCMS. Cross-linkingof /3-Lactoglobulinby 6. To j3-lactoglobulin (1%w/v) in 0.1 M sodium phosphate (pH 7.4, 15 mL) was added NMDC (1.44 mmol), and the solution was incubated at 37 'C. A similar control without NMDC was also incubated under the same conditions. At 0, 20, 46, 67, 94, and 140 h of incubation 2.5 mL aliquots were removed and frozen a t -70 "C. Covalent cross-linking of ,&lactoglobulinwas evaluated by electrophoresis on SDS containing polyacrylamide gels (10-20% linear gradient) and stained with silver nitrate. Protein samples were boiled for 5 min in sample buffer containing 1.0% sodium dodecyl sulfate (SDS) and 2% dithiothreitol before electrophoresis.
Results Incubation of [13C=S]NMDC. The downfield region of the 13C NMR spectrum of [13C=S]NMDC contained a single resonance at 211.8 ppm corresponding to the
200
190
180
170
160ppm
Figure 2. D o d e l d region of the l3C Fourier transform NMR spectra of [l3C=S1NMDC. All spectra were obtained at 75 MHz in 20 nun sample tubes. (A) [13C=S]NMDC immediately after addition to 0.1 M sodium phosphate buffer (pH 7.5) showing a single resonance (211.8 ppm) for the enriched thiocarbonyl carbon; (B)solution from (A) &r 7 d incubation demonstrating generation of bis(thiocarbamoy1) disulfide (bis-6) (202.2 ppm), 1,3-dimethyl[l3C=Slthiourea 10 (180.2 ppm), and [W]HC03(161.2 ppm); (C) 14 d incubation demonstrating accumulation of thiourea and HC03-; (D) at 21 d signals for the starting dithiocarbamate and its disulfide were no longer detected, and a new signal (163.0 ppm) from 1,3-dimethyl[13C=Olurea (11) was present.
enriched thiocarbonyl carbon (Figure 2A). Incubation of NMDC at 37 "C in neutral aqueous solution gradually produced three new signals at 202.2, 180.2, and 161.2 ppm from genesis of bis(thiocarbamoy1) disulfide (bis-6), thiourea, and HC03-, respectively (Figure 2B). With further incubation there was gradual loss of the signals attributable to dithiocarbamate and bis(thiocarbamoy1) disulfide with an increase in intensity observed for thiourea and HC03- (Figures 2C and 2D). In addition, with longer periods, a signal at 163.0 ppm became evident (Figure 2D) and was assigned to formation of 1,3dimethyl[W=O]urea (11). Extraction of the aqueous solution with ethyl acetate and analysis by G C N S verified the presence of 1,3-dimethylurea and 1,3-dimethylthiourea (10). Incubation of [l3C=S1NMDC in the Presence of Bovine Serum Albumin. Addition of 13C-enriched NMDC produced two signals corresponding to dithiocarbamate (211.8 ppm) and bis(thiocarbamoy1) disulfide (202.2 ppm) in the downfield region of the 13C NMR spectrum of bovine serum albumin in addition to the protein aryl (125-135 ppm) and acyl (172-181 ppm) carbons (Figure 3A). After 3 d of incubation multiple new 13C-enriched resonances were detected with the most intense new signals present in the region of thiourea (178-184 ppm), HC03- (161 ppm), and COZ(127 ppm) absorbance and a relatively intense unidentified signal at 134 ppm (Figure 3B). With continued incubation there was loss of signals for dithiocarbamate, bidthiocarbamoyl) disulfide, and COz with a new resonance present at 121 ppm (Figure 3C). Dialysis and concentration of the incubated protein solution produced the 13C NMR spectrum in Figure 3D showing 13C-enrichedmethylthiourea (178-184 ppm) and methylurea (161 ppm) moieties
Chem. Res. Toxicol., Vol. 8, No. 2, 1995 267
Methyldithiocarbamate Protein Adducts
200
180
160
140
120ppm
Figure 3. 13C NMR spectra demonstrating adduct formation on bovine serum albumin by [13C=S]NMDC.Spectra A-C were obtained at 75 MHz in 20 mm sample tubes, and spectrum D was obtained at 125 MHz in a 10 mm tube. (A) Bovine serum albumin (0.6 mM) in 0.1 M sodium phosphate (pH 7.5) immediately after addition of [13C=S]NMDC (0.25 mmol), demonstrating signals for protein acyl (172-181 ppm) and aryl (125-135 ppm) carbons and I3C-enrichedNMDC (211.8 ppm) and N-methyl bis(thiocarbamoy1)disulfide (202.2 ppm) thiocarbony1 carbons (offset plot is a 4x vertical expansion);(B) protein solution in (A) after 3 d incubation showing generation of multiple 1%-enrichedresonances in the region of dithiocarbamate ester (195-200 ppm), thiourea (178-184 ppm), HC03- (161 ppm), COz (127 ppm), and a relatively intense unidentified signal at 134 ppm; (C) a h r 13 d [lW=S]NMDC or its disulfide was not detected and the signal at 127 ppm was either no longer present or had shifted to 121 ppm; (D)dialysis and lyophilization of the solution in ( C ) has removed the 13C-enrichedsignals not covalently associated with protein, revealing the presence of methyl [W=O]urea adducts (162 ppm) in addition to [l3C=S1thiourea moieties (178-184 ppm).
present on the protein which were verified using complete acid hydrolysis followed by GC/MS. Incubation of S-Methyl N-Methyl[l3C=S1dithiocarbamate in the Presence of Bovine Serum Albumin. The 13C-enriched thiocarbonyl carbon of 7a gave rise to two 13CNMR signals (200.0and 203.3ppm), as a result of geometric isomerism (7, 151,in addition to the unenriched signals originating from bovine serum albumin (Figure 4A). Upon incubation at 37 "C new signals were observed corresponding to thiourea moieties (178184 ppm), HC03- (161ppm), and COZ(126ppm) with a relatively intense unidentified signal at 121 ppm (Figure 4B). With longer incubation times there was continued production of thiourea, HC03-, and COz (Figure 4C). Dialysis and concentration of the protein solution demonstrated the presence of thiourea (178-184 ppm) and urea (161-162 ppm) moieties on the protein. Methylthiourea Formation on the a-Amino Groups of Terminal Leucine Residues of p-Lactoglobulin. The total ion mass spectrum of synthetic leucine methylthiohydantoin (3b)contained major peaks at 186,143,and 130 (Figure 5A). Incubation of p-lactoglobulin with [13C=S]NMDC resulted in the formation of methylthiourea derivatization on the amino groups of the N-terminal leucine residue. Leucine methyl[ 13C=S]thiohydantoin was obtained from the derivatized P-lactoglobulin using limited hydrolysis. The total ion mass spectrum of the labeled 3b obtained from derivatized P-lactoglobulin exhibited major peaks a t 187, 144,and
200
180
160
140
120ppm
Figure 4. 13C NMR spectra demonstrating adduct formation on bovine serum albumin by S-methylN-methyl[13C=Sldithiocarbamate. Spectra A-C were obtained at 75 MHz in 20 mm sample tubes, and spectrum D was obtained at 125 MHz in a 10 mm tube. (A) Bovine serum albumin (0.6 mM) in 0.1 M sodium phosphate (pH 7.5) immediately after addition of 13C labeled ester (0.25 mmol) showing two 13C-enrichedsignals for the thiocarbonyl carbon (203.3 and 200.0 ppm); (B)after 7 d incubation new signals attributable to 13C-enrichedN-methyldithiocarbamate (212 ppm), thiourea (178-184 ppm), HC03(161 ppm), COz (126 ppm), and an unidentified signal at 121 ppm; (C) at 21 d there appears to be a broad signal at the base of the HC03- signal (161-162 ppm) and a decrease in intensity of the signal at 121 ppm; (D)dialysis and lyophilization has removed all W-enriched functional groups not incorporated into the protein and reveals the presence of enriched thiourea (178184 ppm) and urea (161-162 ppm) moieties.
131 (Figure 5B). Leucine methyl[l3C=O1hydantoin (3a) was produced to a lessor extent, and its presence on /l-lactoglobulin was verified by peaks at 171,128,and 115. Following incubation of /3-lactoglobulin with (Nacetyl-S-methylthiocarbamoy1)cysteineboth 3a and 3b could be isolated and identified by GCNS. Ratios of hydantoin to thiohydantoin produced by [l3C=S1NMDC and (N-acetyl-S-methylcarbamoy1)cysteinewere 0.11and 0.14,respectively. Methylthiourea Formation on €-AminoGroups of Lysyl Residues of Bovine Serum Albumin. Synthetic methyl e-lysyl [l3C=S1thiourea was analyzed by GC/MS as its bis(TBDMS) derivative. The 13C-enrichedthiourea lost methylamine on the column to form the corresponding isothiocyanate with major peaks at 360,332,and 258 resulting from successive loss of tert-butyl, tert-butyl and carbonyl, and [(tert-butyldimethylsilyl)oxylcarbonylgroups from isothiocyanate (Figure 6A). The presence of methyl e-lysyl [13C=S]thiourea on bovine serum albumin incubated with methyl[l3C-S]dithiocarbamate was identified by multiple ion monitoring following complete acid hydrolysis of the protein (Figure 6B). Incubation of bovine serum albumin with S-methyl N-methyl[13C=Sldithiocarbamate also generated labeled methylthiourea adducts on +amino groups of lysine that could be identified by GC/MS following hydrolysis. Rates of Methylthiourea Formation on p-Lactoglobulin. The amount of leucine methylthiohydantoin that could be obtained following N-terminal methylthiourea formation on /?-lactoglobulin by NMDC or (Nacetyl-S-methy1thiocarbamoyl)cysteine(7b) was measured. The data are presented in Figure 7 in the form of
258 Chem. Res. Toxicol., Vol. 8, No. 2, 1995
1
Abundance X lo3
400
2oo0
Abundance X 1O3
130
800 -j 600
Valentine et al. 258
120
A A
I
186
90
I
4
8 80
40
120
160
200
332
100
400
B
B
100
320
240
58
31
120
160
360 144
80 60 43
L.I_
57
40
20 0 40
70
187
100
130
160
l
4
d z
scatter plots as nmol of adductJmg of protein against time. The relative rates of adduct formation are approximated by the slope of each curve, 0.215 and 0.032 for 6 and 7b, respectively. Cross-Linkingof /?-Lactoglobulinby NMDC. Incubation of ,&lactoglobulin (-18 K) with NMDC produced intermolecular cross-linking as indicated by dimer formation at 48 and 72 h demonstrated by SDS-polyacrylamide gel electrophoresis (Figure 8). With longer incubation (96 h) higher molecular weight species were detected. Control samples, treated identically except for the absence of NMDC, did not demonstrate the generation of higher molecular weight species relative to monomeric P-lactoglobulin.
l
l 20I ' 280
260
190
Figure 5. Identification of methylthiourea adducts on the a-amino groups of N-terminal leucine in j3-lactoglobulin by thiohydantoin formation. (A) Total ion mass spectrum of authentic leucine methylthiohydantoin (3b)with major peaks at 186,143,and 130;(B) the total ion mass spectrum of the 13C labeled thiohydantoin prepared from j3-lactoglobulin derivatized using [13C=SlNMDCwith major peaks at 187,144,and 131.
I
332
300
320
340
360
d Z
Figure 6. Identification of methylthiourea adducts on the €-amino groups of lysyl residues in bovine serum albumin. Following total acid hydrolysis of bovine serum albumin de-
rivatized with [13C=S]NMDCthe hydrolysates were derivatized (MTBSFA + 1%TBDMCS in acetonitrile)and analyzed by GC/ MS. (A) Total ion mass spectrum of authentic E-lysyl N-methyl[13C=Slthiourea(4) showing decomposition to lysyl-~-[13C=S]isothiocyanate on the column with major peaks at 360 (M tert-butyl),332 (M - tert-butyl-CO),and 258 (M- COaTBDMS); (B) multiple ion mass spectrum of acid hydrolysate obtained from bovine serum albumin incubated with [W=S]NMDC, demonstrating the presence of 4 with l3C label at the thiourea carbon. 20
.-C
8
15
n
F .
5 10 z
V
P
Discussion The decomposition of dithiocarbamates in aqueous systems proceeds through several mechanisms with the individual contributions of each to the overall rate governed in part by the structure of the dithiocarbamate and pH of the medium. Both N-dialkyl and N-monoalkyl un-ionized dithiocarbamic acids undergo proton transfer from sulfur to nitrogen followed by reversible cleavage (12,21,22). As a result, CS2 and parent amine are the major decomposition products when the pH is close to or below the pKa of the dithiocarbamic acid. In basic systems N,Z?-dialkyldithiocarbamatesare relatively stable whereas N-monoalkyldithiocarbamates produce alkyl isothiocyanate and hydrogen sulfide (12, 21). Base catalyzed abstraction of the hydrogen attached to nitrogen a t high pH (21) or proton transfer from nitrogen to sulfur resulting in cleavage (12, 21, 23) are both mech-
0
10
20
30
40
50
60
70
80
time in hours Figure 7. Rates of N-terminal thiourea formation by NMDC and 7b.Thiohydantoin 3b was measured using limited acid hydrolysis and GC/MS following incubation of /?-lactoglobulin with NMDC or (N-acetyl-S-methylthiocarbamoy1)cysteine (NMDC-S-Cys). Derivatization proceeded more rapidly for the free dithiocarbamate.
anisms for the degradation of N-monoalkyldithiocarbamate ions to alkyl isothiocyanate and hydrogen sulfide. Evidence has been presented that inductive effects exerted by the nitrogen substituents may influence the latter mechanism (15);i.e., electron donating substituents enhance the rate of isothiocyanate formation. Additionally, dithiocarbamates are capable of undergoing oxidative coupling with a second dithiocarbamate or sulfhydryl
Chem. Res. Toxicol., Vol. 8, No. 2, 1995 259
MethyldithiocarbamateProtein Adducts
CH3NH2 + CS,
kDa
+
Na’
205 116 97 66
H*H+ C
Ti
S
II
CH3HN-C-S- Nat
CH3HN-C-SR 7
a, R = CHs b, R = CH2CHC02H
45
I
NHCOCH3
29 CH3HN-C-S CH3HN-C-S
II
Figure 8. Intermolecular cross-linking of /I-lactoglobulin by NMDC. Solutions of /I-lactoglobulin (1%w/v) in 0.1 M sodium phosphate buffer (pH 7.5) were incubated at 37 “C and samples obtained at 0, 24,48, 72, and 96 h. Protein samples were run on SDS containing gels in the presence of dithiothreitol and stained using silver nitrate. Lanes 1-5 show /I-lactoglobulin alone (-18K), lanes 6-10 /I-lactoglobulin with 90 mM NMDC. Increasing lane numbers within each group correspond to longer incubation times. Dimer formation by NMDC was evident at 48 h.
resulting in a bis(thiocarbamoy1) disulfide or mixed disulfide, respectively. These disulfides can be reduced back to dithiocarbamates under physiological conditions or may undergo further oxidation to isothiocyanate and sulfur (9, 12,21). At pH 7.5, generation of methyl isothiocyanate appeared to be a predominant route of decomposition for NMDC as evidenced by the accumulation of 1,3-dimethylthiourea (10)(Figure 2). Production of methyl isothiocyanate 8 may have occurred both through intramolecular proton transfer with elimination of HS- and by oxidation of bis(thiocarbamoy1) disulfide (bis-6). Although generation of methylamine, necessary for the production of 1,3-dimethylthiourea, can occur through elimination of CS2 by 6, the absence of a signal for [13C]CS2 and the slow rate of cross-linking observed for /3-lactoglobulin suggest other mechanisms may have contributed. Another potential source of amine involves hydrolysis of methyl isothiocyanate to yield methylamine and COa through the intermediacy of methyl isocyanate (9). Evidence for hydrolysis by this route was provided by detection of 13C-enrichedHC03- and 1,3-dimethylurea (11)(see Figure 9). Isothiocyanates are “soft” electrophiles and may react spontaneously with “soft” nucleophiles such as sulfhydryl groups of glutathione or proteins resulting in formation of dithiocarbamate esters (24). Although less thermodynamically favored, the more basic “hard” NH nucleophilic centers of proteins may also add to isothiocyanate yielding thiourea adducts. The formation of dithiocarbamate esters is reversible under physiological conditions whereas thiourea formation is not. Accordingly, when the fate of protein isothiocyanate adducts was monitored as a function of time, there was a rapid formation of dithiocarbamate esters accompanied by a more gradual accumulation of thiourea structures (15). Eventually, the stability of the thiourea and greater number of amine groups resulted in the consumption of all available isothiocyanate to the exclusion of dithiocarbamate ester. Similarly, in this study protein S-linked NMDC esters coexisted with N-methylthiourea adducts initially but were gradually converted to thiourea and urea adducts. As with 6 alone evidence for isothiocyanate and possibly
II
I
\
CH3HN-C-NHCH3 10
II
CH3HN-C-NHCH3 11
CH3NH2
+
co2
Figure 9. Decomposition of methyldithiocarbamate and methyldithiocarbamate esters at physiological pH. Methyldithiocarbamate (6) may undergo cleavage to parent amine and CS2. Alternatively, methyl isothiocyanate (8) may be generated directly by elimination of HS- from 6 or through oxidation of 6 to bis-6. Once formed, 8 may add methylamine generated from cleavage of 6 or hydrolysis of 9 to form NJV-dimethylthiourea (10).Additionally, the detection of NJV-dimethylurea (11)and bicarbonate also supports generation of methyl isocyanate 9 from 8 under these conditions.
dithiocarbamate ester hydrolysis was afforded by the generation of 13C-enrichedCOS, HCOS-, and N-methylurea adducts. It is recognized that not all phase I1 conjugation reactions are detoxifj.ing in nature and that these conjugated forms may serve to protect reactive species and distribute them to target organs (24, 25). For example, conjugation of methyl isocyanate with glutathione as an S-linked thiocarbamate ester has been proposed as a mechanism for distributing the reactive methyl isocyanate to multiple organs following inhalation exposure, and the reversibility of thiocarbamate ester formation has been verified in model systems (26, 27). In vivo experiments have shown that conjugation of 8 to glutathione is a quantitatively important route of biotransformation and that direct conjugation of NMDC to glutathione appears to be catalyzed by glutathione transferase (7). Examination of dithiocarbamate esters in the present study demonstrated that these conjugates may be capable of acting as a molecular shuttle for transport of the acylating species to critical targets. In addition, these data suggest that dithiocarbamate esters can serve as a source for isocyanate. Generation of isothiocyanate under physiological conditions has been demonstrated previously for monoalkyldithiocarbamates on the +amino group of lysine (15,28, 29). In an aqueous solution in the absence of free sulfhydryl or amino groups the stability of the isothiocyanate generated was sufficient to allow observation by 13C NMR. In the protein solutions examined here, identification of methyl isothiocyanate was most likely precluded due to a combination of its rapid reaction with abundant protein nucleophiles, similar chemical shifts of the protein aryl carbon resonances, and the characteristically broad signal associated with isothiocyanates due to quadrupolar broadening (30).Among the signals observed corresponding to other decomposition products, two require f&her investigation in order to determine
260 Chem. Res. Toxicol., Vol. 8, No. 2, 1995
their identity. One, occurring at 121 ppm, has the same chemical shift as methyl isocyanate (311,but it is unlikely that this signal was produced by methyl isocyanate given its short half-life in aqueous solution (32). Alternatively, sequestration of 113ClC02,which usually absorbs a t 125.9 ppm in water (311, into a hydrophobic region of bovine serum albumin may have resulted in an upfield shift accounting for the signal at 121 ppm. The chemical shift of the second unidentified signal, 134 ppm, suggests that thiocyanate ion (31j may be produced in the decomposition of NMDC in the presence of protein. Neither the signal at 121 nor that at 134 ppm appeared to be covalently associated with the protein. Covalent cross-linking of proteins from evolution of CS2 has been demonstrated previously for Nfl-diethyldithiocarbamate (33). In those experiments considerable dimer (-36K) and trimer (-54K) formation was evident after 24 h, whereas 2-4 d were required for dimer formation by NMDC, despite the higher concentration employed. Two processes may have contributed to the lower rate of cross-linking observed for NMDC. In the first, slower release of CS2 is predicted for NMDC on the basis of the relative rate constants for decomposition of un-ionized Nfl-diethyldithiocarbamate relative to NMDC (34). In the second, production of methylthiourea adducts on protein amino groups may serve to decrease the number of protein sites available to participate in cross-linking. Previous investigations have demonstrated the decomposition of NMDC to produce methyl isothiocyanate, CS2, hydrogen sulfide, and methylamine under experimental conditions (9)and in the environment (2). Studies on the metabolism of NMDC and 8 have shown that they are readily conjugated with reduced glutathione and excreted in the bile or in the urine as a mercapturate (7). It is proposed that, in addition to conjugation with glutathione, acylation of macromolecules by isothiocyanates derived from dithiocarbamates may be a cytotoxic mechanism of these compounds. Further, the comparable cytotoxicitiesreported for isothiocyanates and their glutathione conjugates suggests that the formation of these conjugates is reversible and may serve to deliver isothiocyanates to cellular targets (35, 36). This investigation demonstrates the ability of NMDC and its S-linked dithiocarbamate esters to acylate proteins, resulting in dithiocarbamate ester, thiourea, and urea adducts, with the thiourea and urea exhibiting the greatest stability. Monoalkyldithiocarbamates also appear to be a source for isocyanates, the production of which may actually be enhanced through formation of dithiocarbamate esters. These findings support the concept that monoalkyldithiocarbamates and alkyl isothiocyanates have the potential to exert biological effects through acylation of macromolecules and that dithiocarbamates may serve as a source of CS2 leading to covalent cross-linking of proteins within biological systems.
Acknowledgment. We gratefully acknowledge use of the Duke Magnetic Resonance Center supported in part by NSF, NIH, and NC Biotechnology Grants. This publication was made possible by Grants ES06387 and ES02611 from the National Institute of Environmental Health Sciences, NIH.
References (1) WHO (1988) Dithiocarbamate pesticides, ethylenethiourea, and propylenethiourea: a general introduction. In Environmental Health Criteria, Vol. 78, pp 11-68, World Health Organization, Geneva.
Valentine et al. (2) del Rosaria, A.,Remoy, J., Soliman, V., Dhaliwal, J., Dhoot, J., and Perera, K. (1994) Monitoring for selected degradation products following a spill of vapam into the Sacramento River. J . Environ. Qual. 23, 279-286. (3) Larsson, IC S., Amanda, C., Cekanova, E., and Kjillberg, M. (1976) Studies of teratogenic effects of the dithiocarbamates maneb, mancozeb and propineb. Teratology 14, 171-183. (4) Perocco, P., Santucci, M. A., Campani, A. G., and Forti, G. C. (1989) Toxic and DNA-damaging activities of the fungicides Mancozeb and Thiram (TMTD) on human lymphocytes in vitro. Teratogen. Carcinogen. Mutagen. 9, 75-81. (5) Tripathy, N. R, Majhi, B., Dey, L., and Das, C. C. (1989) Genotoxicity of ziram established through wing, eye and female germ-line mosaic assays and the sex-linked recessive lethal test in D. melamguster. Mutat. Res. 224, 161-169. Rasul, A. R., and Howell, J. M. (1973) Further observations on the response of the peripheral and central nervous system of the rabbit to sodium diethyldithiocarbamate. Acta Neuropathol. 24, 161-173. Lam, W. W., Kim, J. H., Sparks, S. E., Quistad, G. B., and Casida, J. E. (1993) Metabolism in rats and mice of the soil fumigants metham, methyl isothiocyanate, and dazomet. J . Agric. Food Chem. 41, 1497-1502. Ainsworth, S., and Leprowski, W. (1991) Metham-sodium spill shows tankcar safety flaws. Chem. Eng. News July 29, 7-8. Turner, N. J., and Corden, M. E. (1963) Decomposition of N-methyldithiocarbamate in soil. Phytopathology 53,1388-1394. Zheng, G., Kenney, P. M., and Lam, L. R T. (1992) Phenylalkyl isothiocyanate-cysteine conjugates as glutathione S-transferase stimulating agents. J . Med. Chem. 35, 185-188. Eklind, K. I., Morse, M. A.,and Chung, F. L. (1990) Distribution and metabolism of the natural anticarcinogen phenethyl isothiocyanate in A/J mice. Carcinogenesis 11, 2033-2036. Drobnica, L., Kristian, P., and Augustin, J. (1977) The chemistry of the NCS group. In The chemistry of cyanates and their thio derivatives (Patai, S., Ed.) pp 1003-1221, John Wiley and Sons, Chichester. Beauchamp, R. O., Bus, J. S., Popp, J. A.,Boreiko, C. J., and Goldberg, L. (1983) A critical review of the literature on carbon disulfide toxicity. CRC Crit. Rev. Toxicol. 11, 169-278. Davidson, M., and Feinlab, M. (1972) Carbon disulfide poisoning: A review. Am. Heart J. 83, 100-114. Valentine, W. M., Amamath, V., Graham, D. G., and Anthony, D. C. (1992)Covalent cross-linking of proteins by carbon disulfide. Chem. Res. Toxicol. 5, 254-262. Valentine, W. M., Graham, D. G., and Anthony, D. C. (1993) Covalent cross-linking of erythrocyte spectrin in vivo. Toxicol. Appl. Pharmacol. 121, 71-77. Brusewitz, G., Cameron, B. D., Chasseaud, K., Gorler, K., Hawkins, D. R., Koch, H., and Mennicke, W. H. (1977) The metabolism of benzyl isothiocyanate and its cysteine conjugate. Biochem. J . 162, 99-107. Mennicke, W. H., Gorler, K., and Krumbiegel, G. (1983) Metabolism of some naturally occurring isothiocyanates in the rat. Xenobiotica 13, 203-207. Mennicke, W. H., Kral, T., Krumbiegel, G., and Rittman, N. (1987) Determination of N-Acetyl-S-(N-allcylthiocarbamy1)-L-cysteine, A principal metabolite of alkyl isothiocyanates, in rat urine. J . Chromatogr. 414, 19-24. Stepanov, V. M., and Krivtsov, V. F. (1965) 3-Methyl-2-thiohydantoins of amino acids. I. Synthesis and properties of 3-methyl-2-thiohydantoins of some aliphatic amino acids, and phenylalanine, and tyrosine. J. Org. Chem. U.S.S.R. 35, 49-53. Joris, S. J., Aspilla, K. I., and Chakrabarti, C. L. (1970) Decomposition of monoalkyldithiocarbmates. Anal. Chem. 42,647-651. Takami, F., Tokuyama, K., Wakahara, S., and Maeda, T. (1973) Decomposition of dithiocarbamates. VI. The decomposition of N-monosubstituted dithiocarbamic acids in acidic solutions. Chem. Pharm. Bull. 21,594-599. Takami, F., Tokuyama, K., Wakahara, S., and Maeda, T. (1973) Decomposition of dithiocarbamates. VII. The decomposition of N-monosubstituted dithiocarbamaic acid in alkaline solutions. Chem. Pharm. Bull. 21, 1311-1317. Baillie, T. A,,and Slatter, G. J. (1991) Glutathione: A vehicle for the transport of chemically reactive metabolites in vivo. Acc. Chem. Res. 24, 264-270. van Bladeren, P. J. (1988) Formation of toxic metabolites from drugs and other xenobiotics by glutathione conjugation. Trends Pharmacol. Sci. 9, 295-299. Pearson, P. G., Slatter, J. G., Rashed, M. S., Han, D., Grillo, M. P., and Baillie, T. A. (1990) S-(N-Methylcarbamoy1)glutathione: A reactive S-linked metabolite of methyl isocyanate. Biochem. Biophys. Res. Commun. 166, 245-250.
Methyldithiocarbamate Protein Adducts (27) Pearson, P. G., Slatter, J. G., Rashed, M. S., Han, D. H., and Baillie, T. A. (1991) Carbamoylation of peptides and proteins in vitro by S-(N-Methylcarbamoy1)glutathioneand S-(N-Methylcarbamoyl)cysteine, two electrophilic S-linked conjugates of methyl isocyanate. Chem. Res. Toxicol. 4,436-444. (28) Amamath, V., Anthony, D. C., Valentine, W. M., and Graham, D. G. (1991) The molecular mechanism of the carbon disulfide mediated cross-linking of proteins. Chem. Res. Toxicol. 4, 148150. (29) DeCaprio, A. P., Olajas, E. S., Chen, X., Fowke, J. H., Zhu, M., and Bank, S. (1992) Characterization of isothiocyanates, thioureas, and other lysine adduction products in carbon disulfidetreated peptides and protein. Chem. Res. Toxicol. 5, 496-504. (30) Jones, R. G.,and Allen, G. (1982) Carbon-13NMR spectra of a series of para-substituted phenyl isothiocyanates. Org. Magn. Reson. 19, 196-203. (31) Kalinowski, H. O., Berger, S., and Braun, S. (1988)The chemical shift. In Carbon-13NMR Spectroscopy, pp 92-449, John Wiley and Sons. Chichester.
Chem. Res. Toxicol., Vol. 8, No. 2, 1995 261 (32) Brown, W. E., Green, A. H., Cedel, T. E., and Cairns, J. (1987) Biochemistry of protein-isocyanate interactions: a comparison of aryl vs. alkyl isocyanates. Enuiron. Health Perspect. 72,5-11. (33) Valentine, W. M., Amamath, V., Amamath, IC,Rimmele, F., and Graham, D. G. (1995) Carbon disulfide mediated protein crosslinking by N,N-diethyldithiocarbamate. Chem. Res. Toxicol. (in press). (34) Miller, D. M., and Latimer, R. A. (1962) The kinetics of the decomposition and synthesis of some dithiocarbamates. Can.J. Chem. 40,246-255. (35) Temmink, J. H. M., Bruggeman, I. M., and van Bladeren, P. J. (1986) Cytomorphological changes in liver cells exposed to allyl and benzyl isothiocyanate and their cysteine and glutathione conjugates. Arch. Toxicol. 69, 103-110. (36) Bruggeman, I. M., Temmink, J. H. M., and van Bladeren, P. J. (1986)Glutathione- and cysteine-mediated cytotoxicityof allyl and benzyl isothiocyanates. Toxicol. Appl. Pharmacol. 83, 349-359.
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