Chem. Res. Toxicol. 1992,5, 496-504
496
Characterization of Isothiocyanates, Thioureas, and Other Lysine Adduction Products in Carbon Disulfide-Treated Peptides and Protein Anthony P. DeCaprio,*9+** David C. Spink,ils Xi Chen,ll Jay H. Fowke,? Mingshe Zhu,i and Shelton Bank11 Laboratories of Biochemical and Genetic Toxicology and of Organic Analytical Chemistry, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201, and Departments of Environmental Health and Toxicology and of Chemistry, The University at Albany, State University of New York, Albany, New York 12201 Received February 10,1992
Carbon disulfide (CS2) is an industrial solvent used in rayon production and as an organic synthetic precursor. It is also a member of the class of neuropathy-inducing xenobiotics known as the “neurofilament (NF) neurotoxicants”. Current hypotheses propose direct reaction of CS2 with NF lysine t-amine moieties as a step in the mechanism of this neuropathy. In this study, covalent CS2 binding in a lysine-containing dipeptide and in bovine serum albumin (BSA) in vitro was characterized. Dipeptide and BSA, incubated with “CS2,exhibited stable incorporation of radioactivity after removal of unbound CS2 and reincubation in physiological buffer for up to 10 days. In contrast, free thiol levels decreased from a maximum immediately following CS2 exposure to near-base-line levels after 10 days, consistent with time-dependent conversion of initially formed N-substituted dithiocarbamate adducts into secondary products. HPLC/thermospray-MS and H P L C N V photodiode-array analysis of CS2-dipeptide adducts confirmed dithiocarbamate formation and demonstrated their conversion into N-alkylisothiocyanates and, ultimately, N,N’-disubstituted thioureas and ureas. The results of UV spectrophotometry of CS2-treatedBSA were also consistent with loss of dithiocarbamate and appearance of thioureas. Similar time-dependent formation of these products, in addition to N,N’-disubstituted thiuram disulfides, was demonstrated in CS2-treated BSA by means of 13C-NMRspectroscopy. SDSPAGE analysis of adducted protein revealed a discrete, higher mobility band, likely representing a specific intramolecular cross-link. In contrast, no evidence for intermolecular protein crosslinking was obtained. Identical results were obtained with cysteinyl-blocked BSA, indicating the lack of formation of N,S-dialkyldithiocarbamate (dithiourethane) cross-links in these preparations. These findings clarify many chemical aspects of covalent CSJpolypeptide interaction and provide unequivocal evidence for the formation of protein-bound isothiocyanate adducts. A comprehensive direct reaction scheme for this neurotoxicant under physiological conditions is proposed.
Introduction Carbon disulfide (CS2)’ is a volatile,hydrophobic solvent with a long history of industrial usage, primarily in rayon production and as a synthetic precursor for organosulfur pesticides and other compounds (1,2). It is also a known human toxicant with effects on the liver and the cardiovascular and nervous systems (2). The neurotoxic effects of acute, high-level CS2 exposure consist of marked behavioral and psychogenic changes (2). In contrast, * To whom correspondenceshould be addressed at the New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509. + Laboratory of Biochemical and Genetic Toxicology, New York State Department of Health. t Department of Environmental Health and Toxicology, State University of New York. 1 Laboratory of Organic Analytical Chemistry, New York State Department of Health. 11 Department of Chemistry, State University of New York. Abbreviations: NF, neurofilamenk BSA, bovine serum albumin; HPLC,high-performanceliquid chromatography;MS, masa spectrometry; SDS, sodium dodecyl sulfate; PAGE, polyacrylamidegel electrophoresis; NMR, nuclear magnetic resonance;2,5-HD,2,5-hexanedione;IDPN,p,Timinodipropionitrile;TS-MS, thermospray-mass spectrometry; ME, (3mercaptoethanol;GK-dipeptide,Nn-acetylglycine-L-lysinemethyl ester, acetate salt.
chronic exposure generally results in a central and peripheral neuropathy affecting long myelinated nerve fibers, with the pathological hallmark of axonal neurofilament (NF)accumulation (3, 4). Because of these chronic effects, CS2 is a member of the class of neuropathyinducing xenobiotics known as the “neurofilament neurotoxicants”,a chemicallydiverse group which also includes 2,5-hexanedione (2,5-HD), acrylamide, and j3,/3’-iminodipropionitrile (IDPN) (5, 6). Both enzymatic and nonenzymatic transformations of CS2 are believed to underlie the toxicity of this agent in vivo (7,8). Oxidative metabolism and enzymatic desulfurization of CS2 yields COS, COZ,and electrophilic sulfur metabolites which may mediate its hepatotoxicity (2, 7, 8). In contrast, the neurotoxic effects of CS2 may involve direct chemical interaction with neuronal macromolecules (6,8). Early studies revealed the ability of CS2 to add to protein amine and thiol nucleophiles under physiological conditions with formation of dithiocarbamate and trithiocarbonate adducts, respectively (9-12). Amine reaction sites included both protein Na amino termini and lysine e-amine moieties (10). Dithiocarbamates are excellent
0 1992 American Chemical Society oa93-22a~t9212~o5-o4~~~o3.oo/o
Lysine Adducts of Carbon Disulfide
transition metal chelators (13),and the behavioral effects of acute CS2 exposure have been attributed to brain neurotransmitter changes following inhibition of the copper metalloenzyme dopamine @hydroxylase(14). In contrast, the mechanism of chronic CS2-induced neuropathy is less clear, but may involve other lysine adduction products (6, 15).
In vivo experiments in rats exposed to 14CS2or C35S2 demonstrated tissue radioactivity in free, acid-labile,and acid-resistant forms, representing unbound CS2, dithiocarbamate and trithiocarbonate adducts, and unidentified stable reaction products, respectively (16, 17). In vitro studies produced similar results (18). N-Monoalkyldithiocarbamates are only moderately stable under aqueous conditions (19-211, and early workers suggested their in vivo conversion to isothiocyanates via elimination of hydrosulfide ion (11,12). These derivatives could account for the acid-stable tissue binding of CS2 (17,21). Others have questioned whether isothiocyanate formation would occur under physiological conditions (6). Alternatively, dithiocarbamates can undergo either reversion back to CS2 and amine or oxidative dimerization to a thiuram disulfide (13,22). These latter derivatives can also break down to isothiocyanates under appropriate reaction conditions (19, 23). Formation of thiuram disulfides and isothiocyanates from CS2-treated W-acetyllysine in aqueous buffer has recently been reported (15), although isothiocyanate formation in lysine-containing polypeptides has not been unequivocally demonstrated. Isothiocyanates are unlikely to be the ultimate stable adduction products in CS2-derivatizedprotein, since they are moderately reactive toward nucleophilic attack by additional free amine and thiol sidechains (21,241. Such reaction would yield N,N'-disubstituted thioureas (6)and N,S-substituted dithiocarbamate esters (dithiourethanes) (15),resulting in intra- and/or intermolecular protein crosslinking. Dithiourethane formation has been reported in bovine serum albumin (BSA) exposed to CS2 at high pH, followed by removal of unreacted CS2 and reincubation in physiological buffer (15). However, dithiourethanes might be expected to undergo either slow exchange with free protein amines (13) or reversion back to the isothiocyanate (25,26). The molecular mechanism of action of the NF neurotoxicants is still unknown. It has been hypothesized that these agents derivatize NF protein lysine e-amines that are required for normal interaction of filaments with each other or with other axonal cytoskeletal elements (6,27). Depending upon the specific adducts formed, the consequences of lysine reaction could include changes in protein hydrophobicity or electrostatic charge. These might be expected to induce physicochemicaland/or conformational changes in the NF proteins. Others have suggested that interfilament covalent cross-linking of adducted NFs represents the critical pathogenetic step in these neuropathies (5). As a preliminary to in vivo neurofilament binding studies, the present investigation examines in vitro covalent lysine-CS2 adduction in BSA and in a lysinecontaining dipeptide. Adducts were quantitated and characterized by [WI-radiolabeling, HPLCI thermospray (TS)-MS and HPLC/UV photodiode-array analysis, 13CNMR spectroscopy, and polyacrylamide gel electrophoresis (PAGE). The results provide the first definitive evidence for isothiocyanate, thiourea, and, additionally, urea formation in CSz-treated polypeptides. A compre-
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 497
hensive direct reaction scheme for this neurotoxicant under physiological conditions is also proposed.
Experimental Procedures Chemicals. Caution: Carbon disulfide is a toxic and highly flammable solvent. It should be handled only under a fume hood with adequate skin protection. BSA monomer, cysteinylBSA, and Nu-acetyl-L-lysine methyl ester were from Sigma Chemical Co. (St. Louis, MO). WS2(specificactivity 15.6 mCi/ mmol) was prepared by Chemsyn Science Laboratories (Lenexa, KS) and was diluted with unlabeled CSz (ACS-grade reagent, Fisher Scientific,Rochester, NY) and methanol to obtain working solutions containing 10% (v/v) CS2 in solvent. Specific activities of 14CS2dilutions were determined by first measuring total CS2 by means of a modified Viles reagent (28), followed by liquid scintillation counting of aliquots of the trapped CS2 to determine radioactivity. Efficiency of counting was determined by using [14C]toluene as an internal standard. W S 2 (99% isotopic enrichment) was obtained from Cambridge Isotope Laboratories (Woburn, MA) and was used as a 10% solution in methanol. Nu-Acetylglycine-L-lysine methyl ester, acetate salt (GK-dipeptide), wae from Bachem Bioscience (Philadelphia, PA). Gadopentetate dimeglumine (Magnevist) was obtained from Berlex Laboratories (Wayne, NJ), and N-butylisothiocyanate was from Aldrich Chemical Co. (Milwaukee, WI). The barium salt of N u acetyl-Nc-dithiocarbamyldysinewas synthesized by a published method (29). In Vitro Incubations. BSA (5 mg/mL, 0.076 mM, in 200 mM sodium phosphate buffer, 0.01% NaN3, pH 7.4) was incubated with 32 mM [14C]-labeled,[W]-labeled, or unlabeled CS2 (10% v/v in methanol) in capped tubes with no head space present, at 37 "C for 0-16 h. Unreacted CS2 was removed by exhaustive dialysis with fresh buffer. The inclusion of 0.01 % azide was to prevent microbial contamination and was found to have no effect on any CS2-BSA reaction parameter. Following dialysis, samples were concentrated approximately 10-fold (to -0.76 mM BSA) by using Centriflo ultrafiltration membrane cones (Amicon, Danvers, MA) and were reincubated at 37 "C for various periods of time (0-672 h). Selected samples were concentrated 20-fold (to -1.5 mM) prior to reincubation. GKdipeptide samples (50 mg/mL in 200 mM sodium phosphate buffer, 0.01 % NaN3, pH 7.4) were incubated at 37 OC in screwcapped tubes for 16 h with 32 mM [W]-labeled or unlabeled CS2 (10% solution in methanol). Unreacted CS2 was removed by five cycles of alternating vacuum (20 mmHg)/argon treatment, followed by argon sparging for an additional 20 min. Samples were then reincubated at 37 "C for various periods of time (0-672 h) and subjected to additional vacuum treatment to remove any regenerated CS2 prior to each assay procedure. HPLC Analysis of GK-Dipeptide Adducts. A t various reincubation times, samples of CS2-treated GK-dipeptide reaction products were separated by reversed-phase HPLC on a NovaPak Cle column (3.9 X 150 mm, 4-rm particle size; Waters, Inc., Milford, MA). The column was equilibrated in 98% eluent A (100 mM ammonium acetate in H20)and 2 % eluent B (100 mM ammonium acetate in 50% acetonitrile). Products were eluted with a linear gradient of eluent B (20% B at 25 min, 50% B at 35 min, 100% B at 40 min, 2% B at 45 min). For thermosprayMS, HPLC was performed using two Waters Model 510 pumps, an automated gradient controller, Model 490 UV detector, and 740 data module, with manual injection, at a flow rate of 1.2 mL/min. TS-MS was performed with a Vestec (Houston, TX) Model 101 thermospray interface and Hewlett-Packard 5970 quadrupole instrument. The ion-source block was maintained at 310 OC, and the vaporizer probe was 230 "C at the tip. Spectra were recorded over the range m/z 120 to m/z 700. For UV photodiode-array detection, HPLC was performed using two Waters Model 6000A pumps, a WISP 710B autoinjector, 720 system controller, and Model 990 array detector (2-s scanning interval, 3-nm resolution), at a flow rate of 1.4 mL/min. Spectra were obtained over the range of 200-400 nm. For detection of hydrophobic reaction products (i.e., thiuram disulfides), a
498 Chem. Res. Toxicol., Vol. 5, No. 4, 1992
DeCaprio et al. lb 1 i
DAY 6
9.
51
DAY 0 0
2
4
6
8
10
Reincubation Time (days)
Figure 1. Quantitation of total CS2 binding ( 0 )and free thiols in CS2-treatedGK-dipeptide. Pep(dithiocarbamate adducts) (0) tide was incubated with WS2,(see under Experimental Procedures) followed by removal of unreacted material and reincubation in buffer. Radiolabeling(liquid scintillation spectrometry) and thiol content (Ellman's reagent) were measured after various reincubation times. Data represent the mean f SD from 3 determinations. modified linear gradient consisting of 2% eluent A (10 mM ammonium acetate in H20) to 98% B (acetonitrile) over 60 min was employed. W-NMR Analysis of BSA Adducts. 13C-NMR spectra of BSA and WSz-treated BSA samples were acquired on a Varian (Sunnyvale, CA) XL-300 NMR spectrometer operating at 75.43 mHz with 20-kHz spectral width and 20 032 data points using broad-band proton decoupling. The pulse width was 10 bs corresponding to approximately a 40° flip angle. The desired signalto-noise ratio was obtained by using 6000-100 OOO transients depending upon the sample. Gadopentetate dimeglumine was added to reduce the recycle time to -0.5 8. This relaxation agent was found to have no effect on CS2-BSA reaction parameters or chemical shift values. Half-milliliter BSA samples (-50 mg/ mL in buffer containing 10% D20 and 5 mM relaxation agent) were placed in 5-mm NMR tubes maintained at 25 OC. The spectra were calibrated by the methine peak of meglumine at 73 ppm and referenced to TMS. Spectra were transformed using 20-Hz digital broadening. Other Methods. PAGE, with or without sodium dodecyl sulfate (SDS) or 8-mercaptoethanol (ME), was performed as previously described (30); protein detection utilized Coomassie blueor silver stain (Bio-Rad,Inc., RockvilleCentre, NY). Protein content was determined using Bio-Rad protein assay reagent. Free thiols were assayed using Ellman's reagent (31). UV absorption spectra of CSp-treatedBSA were obtained with a Beckman DU-70 spectrophotometer. Difference spectra were recorded vs control BSA as the background sample. Samples for liquid scintillation counting were prepared in Aquasol (Du Pont/NEN, Boston, MA) and counted in a Packard Tri-Carb liquid scintillation spectrometer.
Results Quantitation of CSz Binding to GK-Dipeptide. Total covalent binding of CS2 was compared with dithiocarbamate formation (measured as the increase in free thiol content using Ellman's reagent) in 14CS2-treatedGKdipeptide (Figure 1). Following removalof unreacted CS2, samples were reincubated to examine time-dependent changes in these binding parameters. Incorporation of radioactivity indicated reaction of approximately 55% of total lysine under the incubation conditions employed (37 OC, 16 h, saturating concentration of CS2). Total stable binding declined only slightly over the 10-day reincuba-
'''Iu 0
0
31
. 10
20
30
A
~
40
MINUTES Figure 2. Total ion current chromatograms from HPLC/TSMS analysis of CS2-peptide adduction products at various times after removal of unreacted CS2. Products were separated by reversed-phase HPLC in 100 mM ammonium acetate with acetonitrile gradient elution (see Experimental Procedures). TS mass spectra were recorded over the range m/z 120-700. Numbers refer to assigned structures shown in Chart I.
tion period. Adduct levels, as measured by free thiol concentration, were approximately 20 5% lower than those based upon P4Cl-binding immediately following incubation with CSz. In contrast to total CS2 binding, thiol content decreased steadily during reincubation to undetectable levels after 2 days. Characterization of CSz-Lysine Adducts in GKDipeptide. Dipeptide was exposed to CS2, treated to remove unreacted solvent, and then reincubated for various time periods in physiological buffer. Adduction products were separated by reversed-phased HPLC and analyzed by UV photodiode-array detection and TS-MS. Identification was based upon HPLC retention time, UV spectra, and TS-MS characterization of protonated molecules and fragment ions. The structures of GK-dipeptide la and putative CS2-dipeptide adducts are summarized in Chart I. A slow hydrolysis of the methyl ester function of la was noted in the buffer system used, yielding peptide lb and deesterified analogs of various adduction products. This slow hydrolysis occurred whether or not CS2 was present in the incubation mixture and proved to be advantageous for confirming chemical structures of these products. On the basis of chemical considerations and the results of published studies (8-13, 15, 19, 21), three classes of products were anticipated: monoadducted derivatives (Chart I, 2-41, thioureas and ureas (Chart I, 5 and 61, and thiuram disulfides (Chart I, 7). Total ion current chromatograms from HPLC/TS-MS analyses of CS2-adducted dipeptides after 0,2, or 6 days of reincubation are shown in Figure 2. Time-dependent changes in reaction products were apparent. In addition to unreacted peptides lb (1.2 min) and la (3.7 min), two initial products were present, with HPLC retention times of 8.8and 37.3 min. The 8.8-min peak rapidly disappeared, while the latter peak was still present after 2, but not 6, days of reincubation. At 6 days, a number of additional products were noted, and this pattern was not substantially altered after 13 days of reincubation (data not shown). Table I summarizes major TS-MS and UV spectrophotometric data used for structural confirmation of each
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 499
Lysine Adducts of Carbon Disulfide
Chart I. Structures of GK-Dipeptides (la, lb) and Proposed CSz-Dipeptide Adduction Products (2-7)
\ R2
l a : R1 = lb : R' =
OCH,,
R2 = NH,
OH, R2 = NH,
2a: R' 2b: R'
= OCH,, =
OH, R2 = NHCSS'
3a: R' 3b: R'
=
OCH,,
4a: R' 4b:R'
=
= OH,
R2 = NHCSS
OCH,,
= OH,
i"
R2 = NCS R2 = NCS R2 = NCO R2
=
NCO
Sa, 6a: ~1
=
RZ
5b, 6b:R'
=
OCH,,
5c, 6 ~R': = R'
=
OCH,,
x
=
s,o
R2 = OH, X
= OH, X = S,O
=
S,O
7a: R'
=
R2
7b:R'
=
OCH,,
7 ~R': =
Table I. Characterization of CS2-Lysine Adducts in GK-Dipeptide HPLC TS-mass spectra peptide retention mlr (relative intensity) Laax (Chart I) time (min) lb 2b
1.2 2.6
la 2a
3.7 8.8
5c
9.8
5b
22.1
6a
29.7
5a
32.8
3a
37.3
220 220,252, 282 282c(2),274d (2),26W (loo), 228 (11) 220 302 (39),260 (100)-228 (27),203 (1) 220,252, 282 555c (0.2),553" (0.4),288 (2),270 (I), 220 (a),' 235 246 (2).228 (100).189 (0.3) 246" (loo),228 (70),147 (3) b
569 (O.l),547" (O.l),302 (451,288(2), 220 (a), 270 (51,260(43),246 (3),228 (loo), 235 203 (l), 189 (1) 567c (l),5450 (l),286 (8),260 (65), e 228 (loo),187 (20) 583~(l),561" (l),302 (44),260 (loo), 220 (a), 228 (92),203 (1) 235 324~(l),319d (l),302" (loo),270 (2), 240 260 (91,228 (6),203 (10) (broad)
a [M+ HI+. b Not separable from la in ion chromatogram. [M + Na]+. d [M + NHdI+.e Not detectable in HPLC chromatogram.
f s = shoulder.
product. Representative mass spectra are shown in Figure 3. In addition to ions representing the protonated molecules [M + HI+, substantial fragmentation of peptide products was noted, consistent with previous TS-MS studies of peptides (32). Peptide l b exhibited the [M + H]+ ion at mlz 246, in addition to fragments at mlz 228 [M+ H - H20]+ and mlz 147 [M + H -N-acetylglycine]+. Peptide la gave molecular ions at mlz 282 [M + Nal+, mlz 274 [M + NH41+, and mlz 260 [M + HI+, and an mlz 228 fragment [M + H - CH30Hl+ (Figure3A). UV spectra showed a single absorbance maximum at approximately 220 nm for la and lb. On the basis of its UV absorption spectrum, with characteristic maxima of 252 and 282 nm (331, the initial product peak at 8.8 min was assigned to dithiocarbamate 2a. An expected [M + HI+ ion at mlz 336 was not observed for 2a, consistent with previous reports of the thermal lability of dithiocarbamates and their ready conversion to
R2
= OCH, R2 =
OH
= OH
isothiocyanates (i.e., 3a) at elevated temperatures (34). Product 2a exhibited a prominent isothiocyanate ion at mlz 302 [M + H - HzSI+ and fragment ions at mlz 260 [M + H - HzS - CH3COl+ and mlz 228, assigned to [M + H - H2S - CH&O - CH30Hl+ (Figure 3B). A UVdetectable peak at 2.6-min retention time with an absorption spectrum identical to 2a was assigned to structure 2b. A separate ion chromatogram peak for 2b could not be distinguished, due to its incomplete separation from la. The initial product at 37.3-min retention time was assigned to isothiocyanate 3a. An intense [M + H]+ ion at m/z 302 was present, in addition to adduct ions at mlz 324 [M + Na]+ and mlz 319 [M + NH41+. Fragment ions included those present in the spectrum of 2a, in addition to ions at mlz 270 [M + H - CH30Hl+ and mlz 203 [M + H - H2S -N-acetylglycine]+ (Figure 3C). This product exhibited broad UV absorbance at 240 nm, consistent with the reported spectra of alkylisothiocyanatesin polar media (33, 35). A sample of N-butylisothiocyanate in 75% 0.1 M ammonium acetate and 25% acetonitrile exhibited a similar UV spectrum (data not shown). After 6 days, ion chromatogram peaks corresponding to thioureas Sa (32.8 min), 5b (22.1 min), and 5c (9.8 min) were present (Figure 2). These were confirmed on the basis of both expected [M + HI+ ions and characteristic fragments (Table I, Figure 3D). Product 5a exhibited fragment ions at mlz 302,260, and 203, interpreted as the protonated isothiocyanate of GK-dipeptide (Le., [3a + HI+), protonated peptide ([la + HI+), and protonated peptide with loss of N-acetylglycine, respectively. Fragmentation of 5c yielded ions at mlz 288,270,246, and 189, assigned to the protonated isothiocyanate of deesterified dipeptide (i.e., [3b + HI+),protonated isothiocyanatewith loss of HzO ([3b + H - HzOl+), protonated deesterified peptide ([lb + HI+),and protonated deesterified peptide with loss of H2O ([lb + H - HzOl+), respectively. The mass spectrum of thiourea 5b included fragments common to both 5a and 5c. A fragment ion at mlz 228 was present in the spectra of 5a, 5b, and 5c. All three thiourea products
DeCaprio et al.
600 Chem. Res. Toxicol., Vol. 5, No. 4, 1992 260 [M + HI'
0
V 0
V 0
16 hr incubation 6 hr incubation 2 hr incubation
-
24
-
3.0
-20
3 E
0 -
-
1 .o
2'o
v
16
228
- 12
)
"\, 3 E -u 7
2.01 h
J
- 8
2[
.
- 4
(0
z
0
X
Lu 0
z a n z I3
m
a
MASS / CHARGE Figure 3. Representative thermospray-mass spectra of GKdipeptide la (panel A) and CSz-peptide reaction products: (panel B) dithiocarbamate 2a; (panel C) isothiocyanate 3a; (panel D) thiourea Sb. Note expanded scale above mlz 400 in (D); no significant peaks were present above background in the mlz 400700 region in panels A-C. Molecular ions, major fragments, and HPLC retention times are listed in Table I, and fragmentation patterns are interpreted under Results.
exhibited a typically strong UV absorption maximum at 235 nm (33). Peaks assigned to ureas 6a (29.7 min) and 6b (17.2 min) were also observed in total ion chromatograms of 6-day samples. Appropriate molecular ions were present (Table I), and fragmentation patterns analogous to those encountered with thioureas 5a and Sb were apparent. Products 6a and 6b were not present in sufficient concentration to be detected by UV absorbance in HPLC chromatograms. No evidence for formation of products 3b, 4a, 4b, or 6c was obtained. The possible formation of thiuram disulfides 7a, 7b, or 7c, the oxidative dimers of 2a and 2b, could not be addressed by TS-MS due to their marked hydrophobicity and consequent HPLC elution at high acetonitrile concentration. However, some evidence for at least one of these products was obtained using a modified reversedphase gradient and UV photodiode-array detection (see under Experimental Procedures). Under these conditions, a late-eluting peak present in chromatograms of 0-and 2-day reincubation samples exhibited a UV spectrum with maxima at 230, 260, and 285 nm, consistent with the reported spectra of thiuram disulfides (33). Quantitationof CS2 Binding to BSA. The results of binding studies with BSA incubated with 14CS2 (at saturation; -32 mM) for 2, 6, or 16 h, followed by rein-
E. 3
I
Y
%
0
2
4
6
8
10
Reincubation Time (days)
Figure 4. Quantitation of total CS2 binding (solid lines and symbols) and free thiols (dithiocarbamate adducts; dashed lines and hollow symbols) in CSdreated BSA. Protein was incubated (see under Experimental Procedures) with 14CS2for 2 (0,O ) , 6 (v,v),or 16 h (B, a), followed by removal of unreacted material and reincubation in buffer. Radiolabeling (liquid scintillation spectrometry) and thiol content (Ellman's reagent) were measured after various reincubation times. Data represent the mean f SD from 3-5 determinations. cubation in pH 7.4 buffer, are shown in Figure 4. Absolute adduct levels on a mol/mol basis were dependent upon time of exposure and were substantially higher than with the GK-dipeptide (cf. Figure 1). Incorporation of radioactivity was consistent with reaction of approximately 30% (18of 60) of protein amines (aand e) after 16-h exposure. As with the dipeptide, total CS2 binding declined only slightly over the 10-day reincubation period. Although initial free thiol levels were approximately 50% higher on a mol/mol basis than 14CS2 incorporation, a first-order decline in thiol (k = 0.197 dayl) was observed during reincubation. After 10days, residual free thiol was less than 4 mol/mol of protein. UV Spectrophotometry of CS2-TreatedBSA. Spectrophotometric changes were followed in BSA during CS2 treatment (Figure 5A) and after removal of unreacted solvent (Figure 5B). Within minutes of CS2 addition, increased absorbance at 252 and 290 nm was observed, indicativeof rapid dithiocarbamate (and possibly thiuram disulfide) formation (Figure 5A) (33). Further increases were noted at 1h, in addition to a new peak in the 330-nm region. Since no CS2 amine reaction products exhibit significant UV absorbance at 330 nm (331, this peak may have been a reflection of time-dependent conformational alterations in the BSA molecule. Following CS2 removal and reincubation, UV absorbances at 290 and 330 nm gradually decreased to near-base-linelevels. The 252-nm maximum decreased t o a lesser extent, and exhibited a slight shift (to -248 nm). These results are consistent with progressive conversion of initially formed dithiocarbamates to isothiocyanatesand thioureas. Identicalresults were obtained with CS2-treated cysteinyl-BSA,indicating the lack of participation of free thiols in these reactions. W N M R Studies CSz-Treated BSA. In order to determine which CSpdipeptide adduction products identified above might be present in exposed protein, BSA was incubated with 13CS2,followed by removal of unreacted solvent and reincubation in physiological buffer. The identity and time course of formation and loss of various adducts was examined by 13C-NMR. The NMR spectrum of untreated BSA is shown in the bottom panel of Figure 6. Clusters of peaks in the 180 and 130 ppm
Chem. Res. Toxicol., Vol. 5, No.4, 1992 501
Lysine Adducts of Carbon Disulfide 210
230
250
270
290
310
330
350
TU
TU
n
I\
I
ITCh
I B
3 m
4.0
a
I
I
I
I
I
I
-B -
- 4.0
0.0 I
210
230
I
250
1
270
I
290
310
330
350
WAVELENGTH (f"
Figure 5. UV absorbance spectra of CS2-BSA mixtures prior to and following dialysis. Panel A Difference spectra (vscontrol BSA, pH 7.4) recorded at 0 , 2 , 4 , and 60 min following addition of CS2. Dashed line indicates spectrum of pure CS2 in buffer. Panel B: Difference spectra of reincubated CS2-treated BSA (vs control) at 1, 2, and 7 days following dialysis to remove unre-
acted CS2.
regions represent carbonyl (amide and carboxyl) carbons and aromatic ring carbons, respectively. An aqueous solution of the barium salt of the W-dithiocarbamate of Na-acetyllysine exhibited a peak a t 210 ppm (data not shown) corresponding to thiocarbonyl carbon (15). After 1-weekincubation a t 37 "C, apeak at 200 ppm appeared, which was ascribed to the thiocarbonyl carbon of thiuram disulfide. In 13CSz-treatedBSA that was reincubated for 6 h (Figure 6)) new peaks a t 212,203, and 192 ppm were present, assigned to dithiocarbamate (structure 11 in Scheme I), thiuram disulfide 12, and unreacted CSZ (36). The CSZwas probably generated by slow reversion of 11. In addition, the peak at 130 ppm increased significantly, consistent with formation of isothie cyanate 14 (40). A small shoulder observed at 182 ppm indicated early formation of thiourea 17 (41). A very small peak at 198 ppm, possibly representing dithiourethane 18, was present a t this time but not thereafter. Similar incubation of BSA with 12CSzproduced an NMRspectrum identical to that of control, indicating that the new peaks observed in the l3CSZincubation experiments were the result of adduct formation rather than protein conformational alterations associated with lysine binding. Time-dependent changes in the relative intensities of the various adduct peaks were observed with continued reincubation at room temperature. The peaks assigned to dithiocarbamate and thiuram disulfide adducts decreased with time and were not present after 12 days. The peaks assigned to isothiocyanates were more persistent, although they were no longer detectable by 21 days of reincubation. In contrast, peaks assigned to thiourea adducts increased progressively with time and represented
220
200
180
160
140
120
100
PPm
lSC-NMR spectroscopy of CS2-adducte~BSA at various times following removal of unreacted material and reincubation in buffer. Spectrum of untreated BSA is shown at bottom. Peaks are labeled as follows: DTC, N-alkyldithiocarbamate (structure 11 in Scheme I); TDS, NP-dialkylthiuram disulfide (12); TU,NP-dialkylthiourea (17);urea,NP-dialkylurea (16); and ITC, N-alkylisothiocyanate(14). Chemical shifts are relative to TMS. Figure
the major stable product a t the longest time point examined. A new peak at 162 ppm, consistent with the expected chemical shift of an N,N'-disubstituted urea (16) (42), was apparent after 5 days and was present as two singlets after 21 days (Figure 6, top panel). The narrowness of these peaks suggests relatively specific reaction sites for urea formation within the protein. These findings provide clear evidence for similar CSz-lysine adduction pathways in both peptides and native protein. Polyacrylamide Gel Electrophoresisof CSrTreated BSA. Gel electrophoretic studies, with and without SDS and ME, were conducted to examine charge and conformational alterations, in addition to possible covalent crosslinking, in CSZ-treated BSA. Figure 7A illustrates accelerated migration of CSz-treated BSA (lanes 2-5) under nondenaturing PAGE conditions (without SDS or ME) as compared with control protein (lane 1). This enhanced migration was consistent with increased net negative charge and/or a smaller effective radius for the protein. The migration rate did not vary as a function of reincubation time. Enhanced silver staining of protein bands at the 0- and 1-day reincubation time points were also noted, as expected with an increased negative charge (data not shown). Separation of CSz-treated BSA in the presence of both SDS and ME is shown in Figure 7B. The migration of the main band in treated protein (lanes 3-6) was not markedly
DeCapno et al.
602 Chem. Res. Toxicol., Vol. 5, No.4, 1992
-sDs -ME
1 0
Figure 7. PAGE analysis of CSrtreated BSA. Protein was incubated (see under Experimental Procedures) with 32 mM CS2 for 6 h, followed by dialysis and reincubation in fresh buffer. (A) Control BSA (lane 1)and CS2-treated BSA, reincubated for 0, 1,2, or 7 days (lanes 2-5) and separated under nondenaturing conditions (no SDS,ME). (B) Control (lane 2) and treated (lanes 3-6) protein separated under denaturing, reducing conditions (SDSand ME). Time pointssame as in (A). The arrow indicates a new, discrete band in treated protein. (C) Control (lane 2) and treated (lanes 3-6) protein separated under denaturing, nonreducing conditions (SDSwithout ME). Molecular weight markers shown in lanes 1 of (B) and (C) include (top to bottom) phosphorylase B (97.4K), BSA (66.2K), ovalbumin (45.OK), carbonic anhydrase (31.OK), and trypsin inhibitor (21.5K). Marker preparations contained both SDS and ME. Ten percent acrylamide gels with 5 ?6 stacking gel, 2 pg of protein/lane, Coomassie blue staining.
Scheme I. Propoeed Comprehensive Direct (Nonenzymatic) Reaction Scheme for CSZ and Protein under Physiological Conditions &SH
+ CS2
8
%NH2
+ cs,
9
13
14
different from that of control BSA (lane 2). However, a new, slightly faster migrating band became apparent after 24 h of reincubation and increased in concentration with time (Figure 7B, arrow). Comparison with molecular weight markers (Figure 7B, lane 1)indicated a decreased apparent molecular weight of approximately 6K for this new band. Electroelution of this band followed by quantitative amino acid analysis (data not shown) gave a composition indistinguishablefrom control protein, except for a decreased lysine content. This finding indicates that the new band likely represented an intramolecular crosslinked species (i.e., 16,17, or 18) rather than a proteolytic
fragment. In addition, identical resulta were obtained with CS2-treated cysteinyl-BSA, demonstrating that the new protein band was not a reflection of intramolecular crosslinking via 18. No high-MW bands consistent with intermolecular covalent cross-linking were noted in these preparations, even at protein concentrations of up to 100 mg/mL (-1.5 mM). Separation in the presence of SDS but not ME (Figure 7C) revealed a faster migration of both control (lane 2) and CS2-treated (lanes 3-6) BSA as compared with the BSA band present in the MW markers (lane 1). The marker proteins were fully reduced with ME prior to loading on the gel. Under these separation conditions, no clearly resolved, faster migrating band was apparent in CS2-treated samples, although a reincubation time-dependent spreading of the main band was noted.
Discussion The potential mechanistic significanceof direct, covalent modification of protein amines by certain reactive chemical neurotoxicants has been recognized in recent years (5,6, 27). For example, much work has examined lysine €-amine derivatization by the NF neurotoxicant 2,5-HD, which forms substituted pyrrole adducts in NF protein (30).With one exception (40), similar in vivo NF adduction studies with CS2, an agent which induces a neuropathy very similar to that of 2,5-HD (4),are lacking. Little in vitro or in vivo NF protein adduction data are available regarding two other important neurotoxicants, acrylamide and IDPN. Hypotheses have been proposed which postulate common mechanisms of action for these chemically diverse agents (5,6,41). Elucidation of such a mechanism would be a significant step toward the prediction of similar neurotoxic effects for novel or untested compounds. This goal awaits the full characterization of the chemical nature and molecular target sites of these adduction reactions. Based upon the results of the current investigation and previous efforts, a comprehensive reaction pathway for CS2 and protein'under physiological conditions can now be proposed (Scheme I). The chemical interaction between CS2 and biological macromolecules was the subject of much early work ( I , 9, 1I , 12,42),as a consequence of ita widespread use in rayon production and many instances of human poisoning. The formation of trithiocarbonates (10; Scheme I) and dithiocarbamates (11) by addition of CS2 to protein thiols (8) and amines (9) has been known for many years (9,11,12). In contrast, other protein nucleophiles, including hydroxyls and imidazole nitrogens, appear relatively unreactive (9). The known metal ion chelating ability of dithiocarbamates (13,22)led to the hypothesis that they contribute to the neurobehavioral effects of CS2, by means of interference with neuronal enzymes requiring zinc or copper for activity (14). Under conditions of low pH and elevated temperature, formation of dithiocarbamates was shown to be reversible, with consequent release of free CS2 from "acid-labile" tissue forms (28). Early studies also suggested a residual, "acidstable" fraction of CS2 binding (11). More recent work using [14C]- and/or [35S]-labeledCS2 has confirmed the presence of stable adducts in protein exposed in vitro and in vivo (17,18). Without direct experimental evidence, SouEek (11)proposed the formation of isothiocyanate (14) as an alternative breakdown pathway for 11 that could account for acid-resistant protein binding. While this proposal has been reiterated elsewhere and enjoys support based upon chemical studies (7, 17, 21), others have
Lysine Adducts of Carbon Disulfide
questioned whether isothiocyanate formation is likely under physiological conditions (6). No unequivocal evidence for 14 in CSz-adducted protein has been reported, although a recent study demonstrated isothiocyanate formation in CSZ-treatedNu-acetyllysinein aqueous buffer (15).
The present investigation confirms the formation of 11 in CSz-treated polypeptides and provides definitive evidence for its conversion to 14 under physiological conditions. Dithiocarbamates were the initial lysine-CSz reaction products in both GK-dipeptide and BSA, as shown by thiol assay, TS-MS, and UV and 13C-NMRspectroscopy. These adducts disappeared relatively rapidly, while covalently bound radioactivity decreased only slightly over the same period. This finding is consistent with conversion of 11 to secondary adducts, rather than reversion back to CS2 and amine. MS and 13C-NMR spectroscopy results indicated that these secondary products included both 14 and thiuram disulfide 12. Adduct 12 was itself transient, probably undergoing oxidative cleavageto form additional 14 (19).
Isothiocyanate adducts were surprisingly persistent in CS2-treated BSA. Although less reactive than their isocyanate (15) counterparts, these derivatives are still moderately strong electrophiles (24). However, they are relatively hydrophobic and might be expected to associate with nonpolar regions of the protein. This could provide a shielding effect against rapid reaction with additional amine and thiol nucleophiles. Nevertheless, attack by lysine t-amine functions ultimately resulted in the formation of N,N’-disubstituted thioureas (17) in both CS2adducted dipeptide and BSA, as shown by TS-MS and 13C-NMRspectroscopy. Thioureas were still present after prolonged incubation and likely represent a final stable adduction product of CSz and protein. The lack of detection in the present study of substituted dithiocarbamate esters (dithiourethanes; 181, the expected product of nucleophilic attack of free thiols on 14 (21),is riot surprising. Concentration notwithstanding, thiol is favored over amine addition with 14. However, native BSA contains only one free thiol moiety (Cys 341, as compared to 60 potential amine (a and t) reaction sites (43). More importantly, 18 is less stable than 17 under aqueous conditions and would be expected to undergo slow reversion or exchange with protein amines, with ultimate formation of the thiourea (13,25,26). The present findings are in conflict with a recent report of dithiourethane formation in CSz-treated protein (15). That study did not report the presence of 17 (or 14) in BSA exposed to CSz and then reincubated for up to 5 days. One possible explanation involvesthe unphysiologically high pH (- 12) buffer system used by those investigators during the initial CSdprotein incubation. Similar conditions have been reported to result in disulfide cleavage, probably via basecatalyzed hydrolysis, resulting in the generation of additional free thiol reaction sites (44). These would be expected to combine rapidly with 14 to yield 18. The stability of the dithiourethane adduct beyond 5 days was not examined in that study. An unexpected result of the present investigation was the apparent formation of N,N’-disubstituted ureas (16) in both GK-dipeptide and BSA following prolonged reincubation. In one possibility, ureas would be the putative result of the reaction of isocyanates (15) with protein amines (24,451. Although 15 was not detected, its high reactivity would make it, at best, a transient intermediate under the conditions employed. One route to formation
Chem. Res. Toxicol., Vol. 5, No. 4, 1992 503
of 15 from 14 involves base-catalyzed hydrolysis to yield monothiocarbamate 13 ( 2 0 , which could eliminate hydrosulfide to form the isocyanate. Alternatively, direct oxidation of isothiocyanates to isocyanates has been described (45). Other possible schemes do not directly involve the isocyanate. Regardless of mechanism, definitive evidence for urea formation was obtained in the present study, and this derivative represents an additional long-lived adduct in CSz-treated protein. Several CSz-protein adduction products are potential cross-linking structures. Noncovalent cross-linking can occur with 11 by chelation of metal ions (18). Of more significance are the covalent cross-links produced by 12 and 16-18. While 12 and 18 are relatively labile, thioureas and ureas are expected to be quite stable. Thioureabased cross-linking of NFs was considered by Sayre (6) if isothiocyanates could form under physiological conditions. Intermolecular covalent cross-linking of CSz-treated BSA, attributed to 18, has been reported (15). However, the elevated pH conditions used in that study (discussed above) could have also led to dehydroalanine formation followed by lanthionine- or lysinoalanine-mediated crosslinking (46). Despite high protein concentrations in reincubated samples (up to 1.5 mM BSA), no evidence for intermolecular cross-linking was obtained by PAGE under either denaturing or nondenaturing conditions in the present study, indicating that 16 and 17 formation were intramolecular. This lack of thiourea cross-linking between protein molecules may be a reflection of the hydrophobicity of the isothiocyanate, which would preferentially add to buried or shielded amine moieties to yield intramolecular linkages. Intramolecular protein cross-linkingcan be accompanied by an increased migration rate and/or band-spreading in SDS-PAGE, as a result of protein conformational alterations (47). In the presence of both SDS and ME, PAGE separation of CSz-treated BSA revealed a time-dependent increase in a discrete, faster migrating protein band in addition to the main BSA band. Radiolabeling studies of similar preparations indicated total binding levels of approximately 10 mol of CSz/mol of protein, consistent with a maximum of 10 cross-linkslmolecule of protein. It is therefore possible that only certain cross-links induced conformational changes sufficient to affect SDS-PAGE mobility, while the remainder had no effect on this parameter. In contrast, SDS-PAGE separation without ME resulted primarily in slight band-spreading in the direction of increased mobility. Since the conformation of native BSA is already restricted by 17 intramolecular disulfide cross-links (431, the presence of 10 additional thiourea or urea linkages would probably not have had major effects on mobility. Formation of a discrete band, as opposed to simple band broadening, in SDS-PAGE is suggestiveof a highly specific intramolecular cross-linking site. In contrast to the relatively broad 13C-NMRpeak corresponding to 17, the signal from 16 was observed initially as a sharp singlet and ultimately as two singlets. These data are consistent with the presence of one or two unique urea-mediated crosslinking sites, in contrast to a more random distribution of thiourea cross-links. It can be speculated that some undetermined local catalytic mechanism within the protein may operate to enhance formation of 16. The nature and mechanism of specific urea cross-link formation are currently being explored in BSA and other model proteins. The present study has clarified many chemical aspects of covalent CSzfprotein interaction, including the un-
504 Chem. Res. Toxicol., Vol. 5, No. 4,1992
equivocalformation of isothiocyanates under physiological conditions. We provide evidence that N,N’-disubstituted thioureas and ureas are stable protein adduction products of this neurotoxicant, suggesting a possible mechanistic role for covalent cross-linking in CS2 neuropathy. However,since only intramolecular protein crosslinking was observed in this study, the intermolecular linkages necessary for postulated NF-NF cross-linking (5) may not be readily formed. In addition, chemical studies suggest that the relative stability and persistence of structures 11and 14 in protein may be dependent upon a number of parameters, such as the microenvironment of specific lysine side chains, steric and electronic factors, and the local availability of transition-metal cations (19, 21,23). The 11 14 conversion in particular is subject to multiple kinetic influences which are difficult to predict a priori. Consequently, direct toxiciological significance of these monomeric adducts cannot be ruled out, and it is essential to quantitate in vivo levels of all CS2-induced adducts at the putative critical target site, the neurofilament. This is a goal of ongoing research in this laboratory.
-
Acknowledgment. This work was supported by a research grant (38-05172) from the National Institute for Environmental Health Sciences. In addition, support from the National Science Foundation for the purchase of the NMR instrumentation used in this research is gratefully acknowledged. References (1) Brieger, H. (1961) Chronic carbon disulfide poisoning. J. Occup. Med. 3, 302-308. (2) Beauchamp, R. O., Bus, J. S., Popp, J. A., Boreiko, C. J., and Golberg, L. (1984) A critical review of the literature on carbon disulfide toxicity. CRC Crit. Reo. Toxicol. 11, 169-278. (3) Spencer, P. S., and Schaumburg, H. H. (1976) Central-peripheral (4) (5) (6)
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Registry No. la,10236-44-9;lb, 40908-31-4; 2a,141806-340;2b,141806-38-4; 3a,141806-35-1; 5a,141806-36-2; 5b,14180639-5;5c,141806-40-8; 6a,141806-37-3; 6b,141806-41-9; CSz, 7515-0;lysine, 56-87-1.
