Chem. Res. Toxicol. 1995,8, 96-102
96
Carbon Disulfide Mediated Protein Cross-Linking by NJV-Diethyldithiocarbamate William M. Valentine,* Venkataraman Amarnath, Kalyani Amarnath, Fred Rimmele, and Doyle G. Graham Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 Received August 9, 1994@
NJV-Diethyldithiocarbamate and its disulfide are used as pesticides, in industrial processes, and as therapeutic agents, providing numerous opportunities for human exposure. Animal studies and in vitro investigations have demonstrated adverse effects following exposure to dithiocarbamates. The ability of dithiocarbamates to decompose to parent amine and CS2 suggests that these adverse effects may be mediated through release of CS2. The toxicity of CS2 is well established, and covalent cross-linking of proteins has been presented as a potential molecular mechanism of CS2 induced neuropathy. In the present investigation the ability of NJV-diethyldithiocarbamate to effect covalent cross-linking of proteins under physiological conditions is examined. Using 13C NMR, cross-linking was observed to proceed through dithiocarbamate formation on protein amino groups followed by the production of bis(thiocarbamoyl) disulfide, dithiocarbamate ester, and thiourea cross-linking structures. The presence of bis(lysy1) thiourea cross-linking structures was verified by complete protein hydrolysis in conjunction with GC/MS. Generation of inter- and intramolecular cross-linking was established using denaturing polyacrylamide gel electrophoresis under reducing conditions and revealed that cross-linking proceeded more rapidly for Nfl-diethyldithiocarbamate than for equimolar CS2 under similar conditions. Covalent cross-linking of solubilized neurofilament triplet proteins, the putative neurotoxic targets, was examined. Both Nfl-diethyldithiocarbamate and CS2 were able to covalently cross-link the low molecular weight component of the neurofilament triplet proteins, but neither produced intermolecular cross-linking of the medium or high molecular weight component. These results establish that NJV-diethyldithiocarbamate promoted protein cross-linking occurs under physiological conditions and proceeds through liberation of CS2. This process provides a potential molecular mechanism for the toxicity of Nfl-diethyldithiocarbamate and its bis(thiocarbamoy1) disulfide that may contribute to the neurotoxicity of these compounds.
Introduction Salts and disulfides of Nfl-diethyldithiocarbamate (DEDC,l 2; see Scheme 1) are used as insecticides, fungicides, and herbicides, with recent estimates of world consumption on the order of 25 000-35 000 metric tons per year (11. Industrial and agricultural applications include slimicides for the production of sugar, pulp, and paper; accelerators and antioxidants in the vulcanization of rubber; and metal scavengers in the treatment of waste water. Occupational exposures may occur through their use in these various industrial processes or during application to commercial crops. In addition, the general population may also be exposed through residues on food and from medicinal uses. Disulfiram, bis(diethy1thiocarbamoyl) disulfide (l),has been used in alcohol aversion therapy for several decades (2,3). DEDC is presented used or under consideration as a chelator in the treatment of heavy metal intoxication (4, 5 ) and for modulating the toxicity of certain antineoplastic agents (6).DEDC has also generated interest as an immuno-
* Please address correspondence to this author. Phone: 919-6842124;Fax: 919-684-3324; Express Mail: Department of Pathology, Box 3712 M-254, Duke University Medical Center, Durham, NC 27710. Abstract published in Advance ACS Abstracts, November 15,1994. Abbreviations: DEDC, Nfl-diethyldithiocarbamate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; NFL, low molecular weight neurofilament subunit; NFM, medium molecular weight neurofilament subunit; NFH, high molecular weight neurofilament subunit. @
modulator for use in adjunct therapy for the treatment of acquired immunodeficiency syndrome (7,8). Animal studies and in vitro investigations of the toxicity of dithiocarbamates and their bis(thiocarbamoy1)disulfides have shown neurotoxic (9-13),teratogenic (14,15)and mutagenic (14,16-19)effects, and decreased fertility (20, 21 to be associated with exposure to these compounds. Despite the recognized toxicity of these compounds, their underlying mechanisms have not been established, and the interactions of their metabolities with macromolecules have not been completely delineated. A distal sensorimotor neuropathy occurs in susceptible patients receiving disulfiram for alcohol aversion therapy (22,231.The clinical signs and neurofilamentous swellings present in myelinated mons of affected patients (24-26)resemble those produced by subchronic exposure to low levels of CSZin humans (25,27) and animals (28). Given the clinical similarities and that disulfiram is readily reduced in vivo to DEDC that can then decompose to diethylamine and CSZ(2,29),it appears reasonable that disulfiram induced neuropathy may occur through release of CSZ. Its high water solubility and ability to release CS2 gradually may enable DEDC to deliver CSZ into aqueous systems more effectively than direct introduction of CSZ, a compound that is volatile, sparingly soluble in water, and rapidly eliminated from biological systems (30).Previous investigations have demonstrated the ability of CS2 to cross-link proteins intermolecularly as a potential mechanism of neurotoxicity (31-33). In
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Chem. Res. Toxicol., Vol. 8, No. 1, 1995 97
Protein Cross-Linking by N,N-Diethyldithiocarbamate
Scheme 1 s
II
s
S
II
11.
(CH,CH2)2NCS+CN(CH,CH,), 1
AHCCH, It
0
(CH3CHZ)zNCS 2
-
(CH,CH,),NH 3
+
CS,
I
12
CH,C~H
/I
HCi
AH2
0
the present investigation we study the ability of DEDC to produce CS2 and result in covalent cross-linking of proteins under physiological conditions. Model proteins were used to establish the ability of DEDC to covalently cross-link proteins and to determine the relative rates of cross-linking produced by CSz and DEDC in vitro. Specific isotopic labeling in conjunction with 13C NMR and analysis of protein hydrolysates by GC/MS were used to determine the identity of the cross-linking structures produced by DEDC in bovine serum albumin. Additionally, the abilities of DEDC and CS2 to cross-link neurofilament proteins, the putative neurotoxic targets, are compared.
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 (31, 32). Nominal parameters for 13CNMR spectra acquired at 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. Chemical shift assignments were performed as previously described (32). All chemical shifts are referenced to external aqueous 3-(trimethylsilyl)-l-propanesulfonic acid and adjusted to TMS scale by subtraction of 1.7 ppm with an experimental error of fl ppm. A Hewlett-Packard 5890 Series I1 gas chromatograph (GC) connected to a 5971A mass selective detector was used for GC/ MS analyses. The column (HP-5; 25 m x 0.33 mm x 0.25pM) was used with temperature programming.
13
iH2
NH,
14
lysine
Chemicals. Caution: Carbon disulfide is volatile, flammable, irritating to the skin, and toxic;gloves and a fume hood should be used when handling this compound. [WICarbon disulfide (99%) was acquired from Cambridge Isotope Laboratories (Cambridge, MA). Bovine serum albumin fraction V (9699%) and &lactoglobulin were obtained from Sigma Chemical Co. (St. Louis, MO). All the other chemicals were from Janssen Chimica (New Brunswick, NJ). SodiumN~-Diethyl[lSCldithiocarbamate. To a solution of diethylamine (60pL, 0.6 mmol) in water (3mL, cooled in ice) was added [13C]CS~ (30pL, 0.5 "01). The mixture was stirred for 5 h during which time 0.5 N NaOH (1 mL) was added in small portions. The progress of the reaction was followed by UV scan of a diluted solution (5 pL to 3 mL). The pH of the solution was lowered to 7.2 by adding a solution of NaHzP04. Ethanol and most of the water were removed with a Rotovap, and the residue was diluted with phosphate buffer for further studies.
Cross-Linkingof Porcine Neurofilaments by CS2 and DEDC. Porcine neuroflamenta were isolated from spinal cords using published methods (34) and separated by fast protein liquid chromatography. Solutions containing one of the neurofilament triplet proteins (0.2 mg/mL) in 0.1 M sodium phosphate (pH 7.5),4 M urea, and 1 mM phenylmethanesulfonyl fluoride were incubated at 37 "C for 24 and 48 h either alone or with 25 mM DEDC or 25 mM CSZ. Samples were then dialyzed against water, lyophilized, boiled for 5 min in sample buffer containing 1.0% SDS and 2% dithiothreitol, and examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE; 3-10% linear gradient and silver staining). Cross-Linkingof p-Lactoglobulinby DEDC and CSa. To B-lactoglobulin (2%w/v) in 0.1 M sodium phosphate (pH 7.5, 1.75mL) was added (1)CS2 (44pmol), (2)DEDC (44pmol), or (3)DEDC (132pmol), and the solutions were incubated at 37 "C. At 0,24,48,and 72 h of incubation 350 pL aliquots were removed and frozen at -70 "C. Covalent cross-linking of
Valentine et al.
98 Chem. Res. Toxicol., Vol. 8, No. 1, 1995 /?-lactoglobulin was evaluated by electrophoresis on SDS containing polyacrylamide gels (10-20% linear gradient) and stained with Coomassie blue. Protein samples were boiled for 5 min in sample buffer containing 1.0% SDS and 2% dithiothreitol before electrophoresis. Monomer and dimer /3-lactoglobulin were quantified by densitometry at 595 nm and the values obtained from three samples averaged (1, 2, and 3 pg total protein). Statistical significance was determined using Student's t test for differences between 25 mM DEDC and 25 mM CS2. A p value of ~ 0 . 0 was 1 taken as the level of significance. Dimer as percentage of dimer plus monomer is presented as means f SE. Cross-Linking of Bovine Serum Albumin by NJVDiethyl[13C]dithiocarbamate. A 12 mL solution containing bovine serum albumin (4%w/v), NJV-diethyl[13Cldithiithiocarbamate (0.3 mmol), 2H20 (8%v/v), and 0.1 M sodium phosphate (pH 7.5) was incubated at 37 "C and monitored by 13C N M R spectroscopy. At the end of incubation the protein was concentrated by ultrafiltration using Centricon-30 filters (Amicon Inc., Beverly, MA) at 6000g for 30 min, and both the filtrate and the concentrate were examined by 13CNMR. The pH of the filtrate was then adjusted to 2 using concentrated HCl. Bis(Na-acetyl-E-lysyl)thiourea(12). Nu-Acetyllysine (1.9 g, 10 mmol) was taken in water (50 mL), and CS2 (360 pL, 6 mmol) was added. The solution was stirred, and 1N NaOH (6 mL) was added in drops over a period of 2 h. The formation of Nu-acetyllysine dithiocarbamate (10)was followed by W scan. When the reaction was complete, the mixture was heated to reflux in an oil bath (110 "C) for 5-6 h (35).When analyzed by W scan, the maxima for the dithiocarbamate at 254 and 283 nm were replaced by a single peak at 236 nm. The reaction mixture was concentrated and the residue was purified on a column of silica (15-25% water in acetonitrile). The fractions were monitored by TLC, and the product was isolated as a thick liquid: 1.2 g; 13CN M R 6 22.98 (CH3CO), 23.44 (C-41, 28.85 (C5), 32.24 (C-3), 45.00 (C-6), 56.16 (C-2), 175.20 (COzH), 176.75 (CH3CO), 179.61 (C=S); FAB MS mlz 419 (M l)+. Di-E-L-lysylthiourea (13). The protected thiourea from the previous experiment was dissolved in 6 N HC1 (5 mL) and heated to reflux in an oil bath (110 "C). When TLC (20%water in acetonitrile) indicated the absence of starting material, the reaction mixture was evaporated. The residue was coevaporated with ethanol when a white precipitate was obtained. The solid was collected by centrifugation: 13CN M R Q 22.50 (C-4), 28.68 (C-5), 30.40 (C-3),44.51(C-6),54.00 (C-2), 174.57 (COzH), 180.29 (C=S).
+
Results Covalent Cross-Linkingof fl-Lactoglobulinby CSZ and DEDC. Incubation of P-lactoglobulin with either CS2 or DEDC produced intermolecular cross-linking as evidenced by a time dependent increase of high molecular weight protein species and decrease in monomeric protein detected using SDS-PAGE (Figure 1). Dimer and trimer bands showed a progressively greater portion of each migrating more rapidly with increasing incubation time; this finding was consistent with the formation of intramolecular cross-links limiting the unfolding of the protein in SDS-dithiothreitol. Control samples treated identically except for the absence of CS2 or DEDC did not demonstrate the presence of high molecular weight protein. Densitometry (Figure 2) verified that crosslinking continued to progress over the entire 72 h of incubation and that DEDC mediated cross-linking was concentration dependent and dimer production proceeded more rapidly for DEDC than for an equimolar concentration of CS2 ( p < 0.01). 13C NlMR of Protein Cross-Linking in Bovine Serum Albumin. Addition of 13C enriched DEDC produced two signals in the downfield region of the 13C
d
5 6 7 1 3
1 2 3 4
9 10 11 12
13 14 15 16
Figure 1. Inter- and intramolecular crosslinking of /?-lactoglobulin by CS2 and DEDC. Solutions of B-lactoglobulin (2% w/v) in 0.1 M phosphate buffer (pH 7.5) were incubated at 37 "C and samples obtained at 0,24,48, and 72 h. Protein samples were run on SDS containing polyacrylamide gels in the presence of dithiothreitol and stained using silver nitrate. Lanes 1-4 show /%lactoglobulinalone, lanes 5-8 P-lactoglobulin with 25 mM CS2, lanes 9-12 /I-lactoglobulin with 25 mM DEDC, and lanes 13-16 /?-lactoglobulinwith 75 mM DEDC (m, monomer; d, dimer). Increasing lane numbers within each group correspond to longer periods of incubation.
E
40
0
E 30
+
L
a E
e
-
20
.c
0 C
10 Q a
E
.-
U
0
10
20
30 4 0 50 hours of incubation
60
70
80
Figure 2. Rate of dimer formation by CS2 and DEDC in B-lactoglobulin. Monomer and dimer /?-ladoglobulinwere quantified by densitometry of Coomassie blue stained gels and the values for three samples (1,2, and 3 pg total protein) averaged. Dimer as percentage of dimer plus monomer is presented as mean fSE: controls, open triangles; 25 mM CS2, closed circles; 25 mM DEDC, closed squares; 75 mM DEDC, open squares. Dimer formation increased with longer incubation periods and higher concentrations of DEDC. In addition, covalent crosslinking proceeded more rapidly for DEDC than for an equimolar concentration of CS2 0, < 0.01).
NMR spectrum of bovine serum albumin in addition to the protein aryl (125-135 ppm) and acyl (172-181 ppm) carbons (Figure 3A). The more intense signal at 206 ppm corresponds to the enriched thiocarbonyl carbon of DEDC (2), and the smaller signal at 193 ppm corresponds to the thiocarbonyl carbons of disulfiram (1).After incubation for 48 h, four new signals attributable to thiocarbonyl carbons of dithiocarbamates on bovine serum albumin amino terminal aspartate (215 ppm) and lysyl +amines (5,211 ppm), bis(thiocarbamoy1)disulfides (6,201 ppm, and 1,193 ppm), and dithiocarbamate ester (7,196 ppm) were present (Figure 3B). Continued incubation resulted in the loss of DEDC and disulfiram thiocarbonyl resonances and a gradual production of 13Cenriched thiourea functions (11)evidenced by a broad signal centered at 180 ppm (Figure 3C,D). Analysis of the filtrate obtained from the protein solution (Figure 3F) demonstrated that the signal at 161ppm was not covalently associated with modified protein (Figure 3E). The chemical shiR and acid lability (Figure 3G) of this signal are consistent with the production of bicarbonate. Synthesis of ~Lysyl Thiourea. In order to establish the structure of the thiourea cross-link, di-6-lysylthiourea (13)was prepared starting with Nu-acetyllysine. Bis(Na-
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 99
Protein Cross-Linking by NJV-Diethyldithiocarbamate
I
193
J U 1
C
D D
180
,U,U1 200
220
1
'
"
~
~
~
'
~
x 140
8
k(, l
[l3C1thiourea (Figure 5A) after derivatization and GC indicated that decomposition to ~-ly~yl[~~CIisothi~anate occurred on the column, with major peaks at 360 (M 57), 332 (M- 85), and 258 (M- 159). Multiple ion mass spectra were obtained for acid hydrolysates of bovine serum albumin and bovine serum albumin incubated with [13C]DEDC. Thiourea cross-links between lysyl residues in bovine serum albumin incubated with DEDC were identified by the presence of the three major peaks obtained for di-c-lysyl[13C]thiourea(Figure 5B)whereas only background levels of these three peaks were detected in native bovine serum albumin (Figure 5C). Covalent Cross-Linking of Neurofilament Subunits by CSa and DEDC. Purified low molecular weight neurofilament protein (NFL)produced a single band that migrated with an apparent molecular weight of -70K on SDS-PAGE before and after incubation (Figure 6). Adding either CS2 or DEDC to NFL before incubation resulted in the generation of high molecular weight species. Purified medium molecular component (NFM) and high molecular weight component, NFH, did not exhibit the production of any new bands when incubated alone or with CS2 or DEDC.
'
~
120ppm
F
~
220 200 180 160 140 120 PPm
~ ~ ' " ~" I ~' " '1 ' ~ " ~' " l " "' ~ ~1 ' '
220 200 180 160 140 120
Ppm
Figure 3. Low-field region of 13C Fourier transform NMFt spectra showing [WIDEDC mediated cross-linking of bovine serum albumin. Spectra A-D were obtained at 75 MHz in 20 mm sample tubes, and spectra E-G were obtained at 125 MHz in 10 mm sample tubes. (A) Bovine serum albumin (0.6 mM) in 0.1 M sodium phosphate (pH 7.5) immediately after addition of [13C]DEDC(0.3 m o l ) , demonstratingsignals for protein acyl (172-181 ppm) and aryl (125-135 ppm) carbons and 13C enriched DEDC (206 ppm) and disulfiram (193 ppm) thiocarbony1 carbons (offset plot is a 5 x vertical expansion);(B) protein solution from (A) after 48 h incubation showing generation of amino terminal a-dithiocarbamates(215 ppm), N-E-lysyldithiocarbamates (5,211 ppm), lysylthiocarbamoyl disulfides (6,201 ppm), dithiocarbamate ester (7, 196 ppm), thiourea (11,178184 ppm), and bicarbonate (161 ppm); (C) at 96 h incubation thiourea and bicarbonate have increased in intensity; (D) at 7 d incubation signals for DEDC and disulfiram are no longer present; (E) spectrum of (D) after centrifugal concentration; (F) spectrum of filtrate obtained from (D) containing signal at 161 ppm; (G) filtrate at pH 2.
acety1lysyl)thiourea (12)was obtained by thermal decomposition of the dithiocarbamate 10 and purified by flash chromatography. The synthesis was repeated with [13ClCsZ,and the 13C"IR spectrum of bisWKacetyllysy1)[13C]thioureaexhibited a large broad signal at 178.7 ppm corresponding to the enriched thiocarbonyl carbon (Figure 4A). The signal at 161.7 ppm is the result of a small amount of bis(Na-acetyllysyl)[13Clurea also produced during the synthesis. After submitting bis(Na-acetyllysy1)[l3C1thioureato protein acid hydrolysis conditions (6 N HC1,llO "C, 20 h) and partial removal of HC1 by vacuum, the spectrum in Figure 4B was obtained. The Na-acetyl function has been removed, and the carboxyl acyl signal is shifted upfield 7 ppm due to protonation. The enriched thiourea thiocarbonyl and urea acyl carbon signals exhibited paramagnetic shifts of 1.1 and 0.7 ppm, respectively, at this pH. Although the majority of the thiourea has withstood the hydrolytic conditions, some hydrolysis did occur, resulting in the genesis of more 13C enriched urea. Identification of Cross-J-4 Structures by GC/ MS. The total ion mass spectrum of di-c-lysyl-
Discussion In contrast to monoalkyl dithiocarbamates (36-38) only two major products result from the decomposition of dialkyl dithiocarbamates in neutral aqueous solution (36, 37, 391, CS2 and parent amine. The proposed mechanism for decomposition is through unimolecular cleavage of un-ionized dithiocarbamate, with the rate limiting step being the transfer of hydrogen from sulfur to nitrogen (15)(37,39). Electron donating Substituents are expected to aid transfer toward nitrogen, as is the presence of dialkyl substituents on nitrogen, restricting free rotation about the N - C bond, favoring an orientation in which the S-H distance is near optimal for transfer. Thus the net result of adding a second alkyl substituent to nitrogen is that steric and electronic effects are additive, resulting in a 100-fold increase in the observed rate constant for decomposition of un-ionized DEDC compared to that of N-ethyldithiocarbamate (39).
S 15
Incubation of DEDC with protein results in the production of inter- and intramolecular covalent crosslinking. The cross-linking process was observed to proceed through dithiocarbamate (5) formation on amine groups of protein (4) followed by the generation of bis(thiocarbamoyl) disulfide (6), dithiocarbamate ester (71, and thiourea cross-links (11). With longer incubation times there was a diminished contribution from the disulfide and dithiocarbamate ester structures and a gradual accumulation of thiourea due to the stability of the latter structure. The intermediate and cross-linking structures identified for DEDC correspond to those previously reported for CSZ(31,32,40),indicating that DEDC mediated cross-linking of proteins is propagated through the evolution of CS2.
Valentine et al.
100 Chem. Res. Toxicol., Vol. 8, No. I , 1995 Abundance A
A
1258 1200000
332
3 60
8OOOoO
B
400000 188
184
180
176
172
168
164 ppm
Figure 4. l3C NMR spectra demonstrating the acid stability of dily~yl[~3C]thiourea crosslinking structures. (A) Spectrum obtained at pH 7.5 of bis(N~-acetyl-~-lysyl)[~3C]thiourea exhibiting signals for the nonenriched acetyl (174.3 ppm) and carboxyl (180.3ppm) acyl carbons and the enriched thiourea thiocarbonyl (178.7ppm) and acyl urea (161.7ppm) carbons. (B) Spectrum obtained at pH 1 after heating (110"C) sample from (A) in 6 N HCl and partial removal of HC1 by vacuum, showingloss of the acetyl acyl carbon, a 7 ppm diamagnetic shift of the unionized carboxyl carbon, and 1.1 and 0.7 ppm paramgnetic shifts of the enriched thiocarbonyl and urea acyl carbons, respectively. On a molar basis DEDC appears to cross-link proteins more rapidly than CS2 in aqueous systems. The ability of DEDC to serve as a time release source of CS2, enabling a sustained rate of derivatization of lysyl amino groups, may result in a more favorable ratio of derivatized to nonderivatized amine functions for cross-linking than occurs after the direct introduction of CS2. In addition, a sustained delivery of CS2 may be more effective in countering the rapid elimination of CS2 in an open system. Further enhancement of cross-linking by DEDC over CS2 was also possible through the increased concentrations obtainable with DEDC due to its greater water solubility. Evidence has been presented previously for the production of urea cross-linking structures by CS2, presumably through the formation of an isocyanate intermediate (40). On the basis of results obtained in dipeptides analyzed by LC/MS, that investigation assigned a signal at 162 ppm in the 13CNMR spectrum of a bovine serum albumin solution incubated with [13ClCS~ to urea crosslinks. The narrow linewidth of the signal at 162 ppm relative to thiourea was attributed to the formation of one or two unique urea mediated cross-linking sites, possibly as a result of a local catalytic effect. In the present investigation a similar signal was observed at 161 ppm for bovine serum albumin incubated with [13C]DEDC. Analysis of this signal demonstrated that it was associated with a lower molecular weight (
