The measurement of 2-thiothiazolidine-4-carboxylic acid as an index of the in vivo release of CS2 by dithiocarbamates

Article abstract of DOI:10.1021/tx960006v

Dithiocarbamates and their disulfides are used extensively as agricultural fungicides, as accelerators of the vulcanization process of rubber in industry, and as therapeutic agents in medicine. The widespread uses of these compounds in agriculture, industry, and medicine provide many avenues of exposure to the human population. Subchronic to chronic exposures to some dithiocarbamates have resulted in the development of neuropathy in humans and experimental animals. Decomposition to CS₂ presents a potential mechanism through which the toxicity of dithiocarbamates may be mediated. The purpose of this study was to determine the potential of dithiocarbamates to release CS₂ in vivo. The ability to release CS₂ was assessed by measuring urinary 2-thiothiazolidine-4-carboxylic acid (TTCA), which is used in industry to measure the exposure of workers to CS₂. In this study, rats were housed individually in metabolic cages and given daily equimolar ip or po doses (1.5 mmol/kg) of N,N-diethyldithiocarbamate (DEDC), disulfiram (DS), N- methyldithiocarbamate (NMDC), or CS₂ for 5 days, and TTCA was measured in urine collected at 24 h intervals. For each compound administered, TTCA was produced in all of the treated animals and the amount of TTCA eliminated in urine from po administration was significantly greater than that from ip administration. The relative rates of TTCA elimination in urine were DS > DEDC ? CS₂ > NMDC for both routes of administration. Following administration of N,N-diethyl[¹³C=S]dithiocarbamate, carbon-13 enrichment at the thiocarbonyl carbon of TTCA was demonstrated using ¹³C NMR. Analysis of urinary TTCA proved to be useful both for establishing the in vive release of CS₂ by dithiocarbamate containing compounds and for evaluating the bioavailability of CS₂. The results appear especially relevant to disulfiram, which is given orally for sustained periods in the treatment of alcoholism and has resulted in the development of neuropathy in susceptible individuals.

Full text of DOI:10.1021/tx960006v

9
10
Chem. Res. Toxicol. 1996, 9, 910-916
Th e Mea su r em en t of 2-Th ioth ia zolid in e-4-ca r boxylic Acid
a s a n In d ex of th e in Vivo Relea se of CS by
2
Dith ioca r ba m a tes
†
‡
‡
‡
D. J . J ohnson, D. G. Graham, V. Amarnath, K. Amarnath, and
,
‡
W. M. Valentine*
Department of Pharmacology, Duke University, Durham, North Carolina 27710,
and Department of Pathology, Vanderbilt University, Nashville, Tennessee 37232
Received J anuary 10, 1996X
Dithiocarbamates and their disulfides are used extensively as agricultural fungicides, as
accelerators of the vulcanization process of rubber in industry, and as therapeutic agents in
medicine. The widespread uses of these compounds in agriculture, industry, and medicine
provide many avenues of exposure to the human population. Subchronic to chronic exposures
to some dithiocarbamates have resulted in the development of neuropathy in humans and
experimental animals. Decomposition to CS
the toxicity of dithiocarbamates may be mediated. The purpose of this study was to determine
the potential of dithiocarbamates to release CS in vivo. The ability to release CS was assessed
by measuring urinary 2-thiothiazolidine-4-carboxylic acid (TTCA), which is used in industry
to measure the exposure of workers to CS . In this study, rats were housed individually in
metabolic cages and given daily equimolar ip or po doses (1.5 mmol/kg) of N,N-diethyldithio-
carbamate (DEDC), disulfiram (DS), N-methyldithiocarbamate (NMDC), or CS for 5 days,
2
presents a potential mechanism through which
2
2
2
2
and TTCA was measured in urine collected at 24 h intervals. For each compound administered,
TTCA was produced in all of the treated animals and the amount of TTCA eliminated in urine
from po administration was significantly greater than that from ip administration. The relative
rates of TTCA elimination in urine were DS > DEDC = CS
2
> NMDC for both routes of
1
3
administration. Following administration of N,N-diethyl[ CdS]dithiocarbamate, carbon-13
1
3
enrichment at the thiocarbonyl carbon of TTCA was demonstrated using C NMR. Analysis
of urinary TTCA proved to be useful both for establishing the in vivo release of CS by
dithiocarbamate containing compounds and for evaluating the bioavailability of CS . The
2
2
results appear especially relevant to disulfiram, which is given orally for sustained periods in
the treatment of alcoholism and has resulted in the development of neuropathy in susceptible
individuals.
sulfiram (DS),1 the bis(thiocarbamoyl) disulfide of di-
ethyldithiocarbamate (DEDC), is used as an aversion
therapy for the treatment of chronic alcoholism (10).
According to WHO (1), the worldwide consumption of
dithiocarbamates in agriculture and industry is between
25 000 and 35 000 metric tons per year. The numerous
and widespread applications in agriculture and industry,
along with the medical uses of these compounds, provide
many avenues through which the human population is
exposed.
In tr od u ction
Dithiocarbamates are used extensively in agriculture
against a broad spectrum of plant diseases caused by
fungi. In industry, dithiocarbamates and their disulfides
are used as accelerators of the vulcanization process of
rubber, as antioxidants in the preparation of plastics, and
as slimicides in water-cooling systems (1). Because of
their strong chelation properties, dithiocarbamates are
used as scavengers in waste-water treatment (1) and in
medicine for the treatment of certain metal poisonings
Human symptoms of acute intoxication from exposure
to dithiocarbamates and their disulfides range from
nausea and ataxia to death, including behavioral symp-
toms such as depression, lethargy, loss of libido, delirium,
and psychoses (11, 12). Dithiocarbamates and their
disulfides have been shown to be neurotoxic (3, 13), and
clinical use of DS has produced a distinct neuropathy
described as a sensorimotor central-peripheral distal
axonopathy with axonal degeneration (12, 14). The
initial symptom of DS neuropathy is usually distal
sensory impairment which is followed by weakness.
These sensory and motor deficits progress proximally
upon continued use of the drug (15, 16).
(
2) and Wilson’s disease (3, 4). Potential adjunctive
oncologic therapies employing dithiocarbamates include
diminishing the toxicity of cisplatin and enhancing the
efficacy of the chemotherapeutic agents, cyclophospha-
mide and bleomycin (3-6). In the area of AIDS research,
dithiocarbamates are being considered because of their
ability to bind the Zn2 -dependent TAT gene product of
HIV, a metal regulatory protein essential for viral
replication (7) and as an immunomodulator (8, 9). Di-
+
*
Author to whom correspondence should be addressed: Dr.
William M. Valentine, Vanderbilt University Medical Center, Depart-
ment of Pathology, B-3318 MCN, Nashville, TN 37232-2561. Voice:
(
615) 343-5836; FAX: (615) 343-5838; e-mail: Bill.Valentine@
1
mcmail.vanderbilt.edu.
Abbreviations: DS, disulfiram; DEDC, N,N-diethyldithiocarbam-
†
Duke University.
Vanderbilt University.
ate; NMDC, N-methyldithiocarbamate; TTCA, 2-thiothiazolidine-4-
carboxylic acid; T
acid.
‡
3
CA, tetrahydro-2-thioxo-2H-1,3-thiazine-4-carboxylic
X
Abstract published in Advance ACS Abstracts, J une 15, 1996.
S0893-228x(96)00006-9 CCC: $12.00 © 1996 American Chemical Society

TTCA as an Index of CS
2
Bioavailability
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 911
Previous investigations have presented several poten-
tial mechanisms to account for the toxicity of dithiocar-
bamates. The majority of these have been based on their
metal-chelating properties. Some studies have attributed
the neurotoxicity of dithiocarbamates to interference with
catecholamine synthesis, resulting from the metal-
chelating abilities of dithiocarbamates (4, 10, 17). By
effectively chelating copper, dithiocarbamates can poten-
tially inhibit dopamine â-hydroxylase (DBH), a copper-
containing enzyme necessary in the biosynthesis of
norepinephrine (11, 17). Similarly, dithiocarbamates
may also inhibit metalloenzymes involved with mito-
chondrial respiration, e.g., cytochrome c oxidase and
monoamine oxidase B, thereby decreasing oxidative
metabolism (7). In addition, the ability of dithiocarbam-
2
+
2+
ates to bind divalent metal ions, including Cu , Zn
,
Cd2 , and Pb , to form a more lipophilic complex which
is readily distributed to the brain, facilitates the ac-
cumulation of these metals in the brain (11). Alterna-
tively, dithiocarbamates may bind essential thiol groups
to form mixed disulfides causing changes in enzyme
conformation, regardless of the metal cofactor (7, 10).
+
2+
A principal decomposition product of dithiocarbamates
in aqueous solution is CS
recognized toxicity of CS , the ability of dithiocarbamates
to form CS rapidly in aqueous solution suggests that CS
may contribute to the toxic effects of dithiocarbamates
14, 15). A previous study has shown that liberation of
CS from DEDC in vitro leads to covalent cross-linking
of proteins (21). Initially, CS reacts with ꢀ-amino groups
on lysyl residues of proteins to form ꢀ-monoalkyldithio-
carbamates followed by decomposition to isothiocyanate;
a second nucleophilic addition by either amino or sulf-
hydryl moieties can occur to form thiourea or dithiocar-
2
(18-20). Considering the
2
2
2
F igu r e 1. The decomposition of dithiocarbamate showing the
(
2
generation of CS and the subsequent reaction with the sulf-
hydryl group on the cysteinyl moiety of GSH to form a trithio-
carbonate that cyclizes to form TTCA. In concept, CS also may
react with the R-NH of free cysteine to form a dithiocarbamate
which cyclizes to TTCA.
2
2
2
2
hood should be used when handling this compound. Diazo-
methane (CH ) is poisonous and explosive. It is handled in
2
N
2
2
bamate ester cross-links, respectively. CS -mediated
polished glassware under a fume hood. Carbon disulfide was
covalent cross-linking in vivo has also been demonstrated
using erythrocyte spectrin (22), and covalent cross-linking
of neurofilament proteins has been proposed as a common
mechanism to account for the identical neuropathies
obtained from EM Science, Gibbstown, NJ . Labeled carbon
disulfide, 13CS
, was purchased from Cambridge Isotope Labo-
2
ratories, Andover, MA. Disulfiram [DS; tetraethylthiuram
disulfide; bis(diethylthiocarbamoyl) disulfide] and corn oil were
obtained from Sigma Chemical Co., St. Louis, MO. N,N-
Diethyldithiocarbamic acid, sodium salt, trihydrate (DEDC),
was obtained from Acros Organics, Pittsburgh, PA. DL-Ho-
mocystine was obtained from Fluka Chemical Corp., Ronkonko-
ma, NY. Methylamine, diethylamine, and Dizald were pur-
chased from Aldrich Chemicals, Milwaukee, WI. Sodium
produced by 2,5-hexanedione and CS
2
(23). Additionally,
the CS produced by dithiocarbamates can react with
2
R-amino groups of amino acids to form a dithiocarbamate
adduct which may give rise to a cyclic structure of the
thiazolidinium type (24). TTCA, which is a thio deriva-
tive of thiazolidine, has been postulated to be formed by
13
N-methyldithiocarbamate (NMDC), sodium N,N-diethyl[ CdS]-
the reaction of CS
2
with the sulfhydryl group of the
dithiocarbamate, and unlabeled TTCA were prepared according
cysteinyl moiety of GSH or cysteine (Figure 1) (25, 26).
to published procedures (21, 30, 31).
TTCA has been shown to be a sensitive indicator of
internal exposure to CS and has become the preferred
2
measurement over the less sensitive and nonspecific
iodine-azide test (27-29).
GC/MS Mea su r em en ts. A Hewlett-Packard 5890 Series II
gas chromatograph connected to a 5971A mass selective detector
was used. An HP-5 column (30 m × 0.25 mm × 0.25 µm) was
used with temperature programming (70 °C for 2 min and 20
°
C/min to 210 °C and hold).
In the present study, the ability of dithiocarbamates
to generate CS
TTCA measurements in rats given equimolar doses of
representative dithiocarbamates or CS over a period of
days. The relative bioavailability of CS generated from
2
in vivo was evaluated using urinary
NMR Mea su r em en ts. A Varian Unity 500 spectrometer
was used. Spectra were acquired at 125 MHz with a sweep
width of 29 000 Hz and a pulse repetition of 1.8 s. Deuterium
oxide was used as an internal lock signal. Chemical shifts were
referenced to external aqueous 3-(trimethylsilyl)-1-propane-
sulfonic acid and adjusted to TMS scale by subtraction of 1.7
ppm.
2
5
2
a dithiocarbamate disulfide, monoalkyldithiocarbamate,
and dialkyldithiocarbamate was compared to the admin-
istration of CS
2
by parenteral and oral routes. In
[13
CdS]-2-Th ioth ia zolid in e-4-ca r boxylic Acid ([13CdS]-
CO (280 mg, 2
(60 µL, 1 mmol)
addition, the origin of the thiocarbonyl carbon in TTCA
TTCA). L-Cystine (240 mg, 1 mmol) and K
mmol) were taken in water (2 mL), and CS
2
3
1
3
was established using N,N-diethyl[ CdS]dithiocarbamate
13
2
and 13C NMR.
was added with stirring. The stirring was continued for 18 h
at room temperature. The reaction mixture was then acidified
to pH 1 with concentrated HCl and extracted with ethyl acetate
Ma ter ia ls a n d Meth od s
(
3 × 5 mL). The extracts were combined, dried over Na
2 4
SO ,
Ch em ica ls. Ca u tion : Carbon disulfide is volatile, flam-
mable, toxic, and a skin irritant; therefore, gloves and a fume
and evaporated. The crude product was crystallized from 1 N
HCl; 80 mg (50%).

9
12 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
J ohnson et al.
In cor p or a tion of [13C]DEDC in to TTCA. [ CdS]DEDC
(2 mmol/kg/day, ip) was given to 3 rats housed in metabolic
cages for 3 days and daily urines were collected. The urine
samples were taken through the assay for TTCA determination
as stated above. Labeled TTCA was determined from the
multiple ion mass spectrum obtained from synthetic dimethyl
13
Tetr a h yd r o-2-th ioxo-2H-1,3-th ia zin e-4-ca r boxylic Acid
(
T
3
CA). Homocystine (6.7 g, 25 mmol) and K
mmol) were mixed together in 40 mL of water, to which CS
2
CO
3
(7 g, 50
(6
2
mL, 100 mmol) was added, and the solution was stirred for 18
h at room temperature. The reaction mixture was acidified to
pH 1 with 6 N HCl and extracted with ethyl acetate (3 × 20
mL). The extracts were combined, dried, and filtered through
Celite and evaporated. The residue was dissolved in 40 mL of
hot 0.5 N HCl, filtered, and cooled to obtain 1 g of white
1
3
[ CdS]TTCA. The pooled methylated extracted samples were
purified by flash chromatography (3:1 n-hexane/ethyl acetate)
and subjected to 13C NMR as previously stated.
o
crystals: mp 168-169 C; UV λmax 283 nm (ꢀ 14 300) and 246
Sta tistica l Com p a r ison s. Statistical analysis for differ-
ences over time were determined by ANOVA. The two-sample
t-test for means was used to determine differences between
treatment groups. A p value of <0.05 was taken as the level of
significance. All analyses were performed using Microsoft Excel
software.
1
3
(
(
8050); C NMR (water) δ 23.2 (C-5), 28.5 (C-6), 56.7 (C-4), 174.3
CO H), and 196.6 (CdS). On methylation with excess diazo-
2
methane, the methyl ester of the 3-methyl derivative was
1
3
obtained quantitatively: C NMR (CDCl
3
) δ 13.3 (N-CH
3
), 22.7
(
1
C-5), 25.72 (C-6), 52.18 (-OCH
72.1 (CdO); MS m/ z 205 (M ) and 146 (M - CO
3
), 59.9 (C-4), 159.0 (CdS), and
+
2
CH
3
).
An im a ls a n d Exp osu r es. Male Sprague-Dawley rats, 200-
50 g, obtained from Charles River Breeding Laboratories
Resu lts
2
Elim in a tion of TTCA in Ur in e. TTCA was present
in the urine of all treated animals for both routes of
administration. TTCA was detected by GC/MS as the
(
Raleigh, NC), were housed individually in metabolic cages,
given finely-ground Purina rodent chow and water ad libitum,
and placed in a room on a diurnal light cycle. Control urine
samples were collected at 24 h intervals for a period of 2-3 days
prior to dithiocarbamate or CS
animal could serve as its own control. Body weights were
determined prior to dosing. The rats were given 1.5 mmol/kg
+
dimethyl derivative of TTCA (M ; m/ z 191) and the
2
administration so that each
fragment at m/ z 132 representing the loss of the car-
bomethoxy side chain. No TTCA was detected in control
urine. The animals showed no visible symptoms of
toxicity or clinical evidence of neuropathy, e.g., hind limb
paralysis, during the course of treatment.
body wt doses of DEDC, DS, NMDC, or CS
(
2
, either ip or po
gavage) once a day for 5 days (n ) 3). This dose was chosen
based on preliminary experiments which had shown this dose
to produce detectable levels of TTCA without signs of overt
toxicity. Urine was collected at room temperature from the
urine collection tubes of the metabolic cages 24 h after each dose
and just prior to administering the next dose. Urine was also
collected at 24, 48, and 72 h after the final dose. The urine
samples were kept at -20 °C until ready for use. Carbon
disulfide was administered in corn oil such that 0.1 mL was
given for each 100 g of body wt. NMDC and DEDC were given
in 0.1 M phosphate buffer, pH 7.5, such that 1 mL of solution
was given for every 100 g of body wt. DS was prepared in 1,2-
propanediol/water (1:2). The suspension (solubility of DS is 0.2
In cor p or a tion of [ CdS]DEDC in to TTCA. 13C-
13
Enriched TTCA was detected by GC/MS as the dimethyl
+
derivative of TTCA (M ; m/ z 192) and the fragment
resulting from loss of the carbomethoxy side chain (m/ z
133) (Figure 2). Figure 3 shows the downfield region of
proton-decoupled 13C NMR spectra corresponding to the
13
methylated urine extracts. The C NMR spectrum
13
obtained from the methylated urine extracts of [ CdS]-
DEDC-treated rats showed the presence of three new
13
resonances. Following the addition of synthetic [ CdS]-
TTCA, there was an increase in the intensity of the
resonance at 170.3 ppm, indicating that this signal
corresponded to the thiocarbonyl carbon of TTCA. The
other two peaks appearing in the spectra remain uni-
dentified. No signal was detected at 170.3 ppm in
extracts of urine obtained from rats administered non-
enriched DEDC.
mg/dL of H
2
O) (32) was sonicated in order to more finely break
up particles to enable passage through a 19-gauge needle or a
gavage tube. Purity of the test compounds was determined
spectrophotometrically to be approximately 99%. The animals
were dosed within 1 h of preparing test solutions. Release of
CS from the dose preparations prior to administration was e4%
2
over a 2 h period.
TTCA Deter m in a tion . Prior to analysis of the test samples,
In tr a p er iton ea l Ad m in istr a tion of Dith ioca r ba m -
a standard curve was generated. Control urine was centrifuged
a tes a n d CS
TTCA elimination over the time course of treatment for
NMDC, DEDC, and CS remained relatively unchanged,
2
. Data obtained show that the mean 24 h
1
0
5 min at 7500 rpm, and then filtered via suction through a
.45 µm filter. Ten milliliter aliquots were spiked with varying
2
3
concentrations of TTCA and a constant concentration of T CA.
whereas the mean 24 h excretion of TTCA from DS
treatment increased (Figure 4). There were significant
differences (p < 0.05) in mean 24 h TTCA elimination
The spiked urines were taken through the entire procedure for
the analysis of TTCA, which was modified from van Doorn et
al. (26). Filtered urine was spiked with a constant concentration
over the course of 5 days when comparing DS with CS
2
,
of T
urine was extracted with ethyl acetate (3 × 2 volumes), and
the extracts were combined and dried over Na SO . The pooled
3
CA and then acidified with concentrated HCl to pH 2. The
NMDC with CS , DEDC with NMDC, and DS with
2
NMDC. Exceptions in statistical significance were found
when comparing the mean 24 h TTCA elimination of
2
4
extracts were taken to dryness by evaporation and then
reconstituted with 2 × 500 µL of methanol; 500 µL of the
methanolic solution was methylated with excess diazomethane,
taken to dryness, and reconstituted in 100 µL of ethyl acetate,
of which 0.5 µL was injected onto the GC/MS and detected, using
multiple ion monitoring, as the dimethyl derivative of TTCA.
DEDC with CS
The relative rate of elimination of TTCA in urine was
DS > DEDC = CS > NMDC. Detectable levels of TTCA
2
(p ) 0.05) and DEDC with DS (p ) 0.14).
2
remained in urine for at least 48 h after cessation of
exposure for all compounds.
Or a l Ad m in istr a tion of Dith ioca r ba m a tes a n d
+
The molecular ion (M ) identified as the dimethyl derivative
was detected at a mass/charge (m/ z) ratio of 191, and the
fragment at m/ z 132 corresponds to the loss of the carbomethoxy
side chain. The concentration of TTCA in the urine was
determined by comparing the ratio of the peak area for TTCA/
CS
2
. In treatment groups where dithiocarbamates and
CS
2
were administered po, the mean 24 h TTCA elimina-
tion from NMDC was relatively constant over the 5-day
treatment period (Figure 5). Among treatment groups,
there were significant differences when comparing the
T
3
CA to a standard curve generated using peak area ratios for
varying concentrations of TTCA and a constant concentration
of T CA. The lower limit of detection for TTCA by this method
was 10 pmol.
mean 24 h TTCA elimination of DS with CS
2
, NMDC
3
with CS , DEDC with DS, DEDC with NMDC, and DS
2

TTCA as an Index of CS
2
Bioavailability
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 913
1
3
F igu r e 3. C NMR spectra demonstrating the incorporation
1
3
13
of CS
2
into the [ CdS]TTCA structure. (A) The downfield
1
3
region of the proton-decoupled C NMR spectrum obtained from
extracted control urine. (B) The C NMR spectrum obtained
from extracted urine of [ CdS]DEDC-treated rats showing the
presence of three new resonances. Synthetic [ CdS]TTCA was
1
3
1
3
1
3
added to the sample and reanalyzed (C). The intensification of
1
3
the resonance at 170.3 ppm following addition of [ CdS]-2-
thiothiazolidine-4-carboxylic acid identifies this signal as the
thiocarbonyl carbon of TTCA. The other two peaks remain
unidentified.
1
3
F igu r e 2. Identification of derivatized [ C]TTCA by GC/MS.
Following derivatization with diazomethane, the urine extracts
were analyzed by GC/MS. The multiple ion mass spectrum of
1
3
derivatized synthetic [ CdS]TTCA (A) shows the molecular ion
M ; m/ z 192) to be the dimethyl derivative. The fragment at
+
(
m/ z 133 represents the loss of the carbomethoxy side chain.
The multiple ion mass spectrum of derivatized extracted urine
1
3
from [ CdS]DEDC-treated rats (B) shows the presence of
1
3
derivatized [ C]TTCA, as indicated by the presence of ions at
m/ z 192 and 133, a difference of one atomic mass unit from
unlabeled derivatized TTCA. The multiple ion mass spectrum
of extracted urine from control rats (C) shows the absence of
TTCA.
with NMDC. Only the comparison of mean 24 h TTCA
elimination between DEDC and CS
2
over the 5-day
treatment period was not statistically different. The
relative rates of TTCA elimination in urine were DS >
DEDC = CS > NMDC. The average 24 h urinary TTCA
elimination was significantly greater from po administra-
tion for all compounds tested than from ip administration
2
F igu r e 4. Urinary TTCA elimination rates obtained from ip
2
administration of DEDC, DS, NMDC, or CS . The first treat-
ment corresponds to day zero, and each urine sample was
collected for 24 h after dosing (n ) 3, mean ( SEM). Days 6
and 7 represent 48 and 72 h after the 5th and final dose.
(Table 1).
Discu ssion
TTCA has been used as a biological monitor of internal
previously used metabolites. Simon et al. (39) deter-
mined that diet could be considered a source of TTCA,
especially a diet rich in brassica vegetables such as
cabbage, from which an extract could yield up to 3 mg of
TTCA/kg. TTCA has also been identified in kidney bean
seedlings treated with a dithiocarbamate fungicide and
in the dwarf pea and in Sakurajima radish as an
endogenous growth inhibitor (39). Dietary sources of
TTCA were not a factor in the present study in that each
animal served as its own control and diet was strictly
regulated. None of the control urines contained TTCA
when analyzed by GC/MS.
exposure for CS in humans (28, 33) and in rats (34, 35)
with the advantage over analysis of free CS in blood of
2
2
being both nonvolatile and detectable in urine. A positive
correlation between exposure level and urinary excretion
of TTCA exists (29, 36, 37), and Kivist o¨ et al. (35) showed
linearity between oral exposures of up to 30 mg/kg CS
and the elimination of TTCA in the urine of rats.
Although only a small percentage of the absorbed CS is
excreted as TTCA, it is readily detectable in urine (35,
8), and TTCA demonstrates greater specificity over
2
2
3

9
14 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
J ohnson et al.
may decompose to regenerate CS
10, 23).
Production of TTCA from administration of dithiocar-
bamates is consistent with previous studies which have
demonstrated the presence of free CS in blood obtained
2
and the parent amine
(
2
from patients receiving DS (40) and in exhaled air of rats
administered DEDC iv (42). The urinary excretion of 3.9
µmol/24 h of TTCA following administration of 1.5 mmol/
kg po CS
.6 µmol/24 h reported for a 1.3 mmol/kg dose of po CS
reported previously (35). The excretion of TTCA follow-
ing oral administration of equimolar doses of CS and
2
measured here is in good agreement with the
3
2
2
DEDC were similar, consonant with a previous in vitro
investigation showing DEDC to be an efficient vehicle
for introducing CS
er levels of TTCA produced by DS relative to an equimo-
lar dose of CS were most likely due to the ability of DS
to be reduced rapidly in vivo to two molecules of DEDC
43). The urinary excretion rate of 2.6 µmol of TTCA/24
h obtained for po administration of 1.5 mmol/kg NMDC
is close to that of 2.3 µmol/24 h reported for 0.4 mmol/kg
po CS (35). The reduced production of TTCA for NMDC
2
relative to DEDC may result from several potential
contributing mechanisms. For one, the observed rate
2
into aqueous systems (21). The high-
F igu r e 5. Urinary TTCA concentrations from rats adminis-
tered DEDC, DS, NMDC, or CS , po. Treatment was started on
day zero, and urine was collected 24 h after administration of
compound just prior to the subsequent dose. Days 6 and 7
represent 48 and 72 h after the 5th and final dose (n ) 3, mean
2
2
(
(
SEM).
Ta ble 1. Mea n 24 h Ur in a r y Excr etion of TTCAa
µmol of TTCA/24 h
2
constants for decomposition to parent amine and CS of
CS2
DEDC
DS
NMDC
dialkyldithiocarbamates are approximately 2 orders of
magnitude greater than those corresponding to analogous
monoalkyldithiocarbamates (19). Additionally, hepato-
toxicity may contribute to diminished TTCA excretion
ip
po
2.24 ( 0.08
3.93 ( 0.19
2.52 ( 0.14
4.00 ( 0.29
2.93 ( 0.31
5.22 ( 0.44
1.16 ( 0.06
2.56 ( 0.27
a
n ) 15; values are expressed as mean ( SE.
(
35) and may be relevant here considering the much
greater cytotoxicity reported for NMDC relative to DEDC
4). Evidence has also been presented to indicate that
The metabolic pathway responsible for the generation
of TTCA has not been established. It has been proposed
(
that TTCA is formed through the addition of CS
sulfhydryl group of the cysteinyl moiety of glutathione
25, 26). Alternatively, addition of CS to the R-amino
2
to the
TTCA excretion parallels hepatic glutathione levels (35)
and NMDC has the potential to reduce free glutathione
levels and consequently TTCA excretion either through
direct enzymatic conjugation (44) or via generation of a
methyl isothiocyanate electrophile (31).
Differences were observed in both the day to day
variability and total amount of TTCA eliminated in urine
between the two routes of administration. When dithio-
(
2
group of cysteine followed by cyclization could also result
in production of TTCA (Figure 1). Support for a role of
glutathione in TTCA generation has been provided in a
previous study that demonstrated urinary excretion rates
of TTCA to parallel levels of hepatic glutathione following
exposure to CS (35). The incorporation of the CS carbon
2 2
into the thiocarbonyl carbon of TTCA observed in the
present investigation is also consistent with the biotrans-
carbamates and CS
2
were given ip, the 24 h excretion of
TTCA into the urine was relatively constant, with the
exception of DS, which appeared to increase with sub-
sequent doses. This apparent increase may have resulted
from accumulation of solid DS in the abdomen due to a
diminished rate of absorption of this insoluble compound
relative to the other test compounds. In contrast, the
urinary TTCA obtained from po administration showed
only NMDC to maintain a relatively constant rate of
TTCA in urine. The other compounds showed significant
variability over the 5-day treatment period and may
reflect a greater variation in absorption by the oral route
of exposure mediated by factors such as the presence of
ingesta. There were significantly greater amounts of
TTCA excreted in urine following po administration
compared to ip injection for each compound. Although
the greater quantities of TTCA obtained for po admin-
istration of dithiocarbamates may have resulted from an
increased contribution of acid-promoted hydrolysis in the
stomach, the difference observed for CS
require further interpretation. Alternatively, quantita-
tive differences in the biotransformation of CS to TTCA
due to route of exposure may be dependent upon effective
dose rates and the resulting levels attained in the liver.
A previous comparison of the uptake rate of CS
excretion of TTCA in urine for gavage and inhalation
exposure showed that the proportion of CS transformed
formation of CS
2
being initiated through the addition of
CS to cysteine or glutathione.
2
A common decomposition product of monoalkyldithio-
carbamates, dialkyldithiocarbamates, and dithiocarbam-
ate disulfides is CS
proposed that CS
2
(Figure 1), and it has been previously
may be the proximate or ultimate
2
toxicant of dithiocarbamates (16, 21, 40, 41). Under
neutral and acidic conditions, monoalkyl- and dialkyl-
dithiocarbamates may undergo acid-promoted decompo-
sition to CS
effects both contribute to increase the rate of decomposi-
tion to CS and parent amine by un-ionized dialkyldithio-
carbamates relative to monoalkyldithiocarbamates (18,
9). Once produced, CS may then react with an ꢀ-amino
2
and the parent amine. Steric and electronic
2
1
2
group of lysine to form a monoalkyldithiocarbamate
which can lead either to the formation of isothiocyanate
-
and SH or to the regeneration of CS
2
and amine (20).
2
appears to
Both monoalkyl- and dialkyldithiocarbamates may also
undergo oxidative dimerization to form a bis(thiocarbam-
oyl) disulfide which in the case of monoalkyl bis(thiocar-
bamoyl) disulfides can oxidize further to isothiocyanate
and sulfur under appropriate reaction conditions (18).
Under physiological conditions, the disulfide is also
readily reduced to yield two dithiocarbamates which
2
2
to
2

TTCA as an Index of CS
2
Bioavailability
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 915
to TTCA decreased as the rate of CS
2
uptake increased
(7) Simonian, J ., Haldar, D., Delmaestro, E., and Trombetta, L. D.
(1992) Effect of disulfiram (DS) on mitochondria from rat hippo-
(35). Consequently, when a considerably smaller ab-
campus: metabolic compartmentation of DS neurotoxicity. Neu-
rochem. Res. 17, 1029-1035.
2
sorbed dose of CS was administered via inhalation over
a 6 h period, greater quantities of TTCA were excreted
than when a bolus dose of CS was administered via
(8) Pompidou, A., Delsaux, M. C., Telvi, L., Mace, B., Coutance, F.,
Falkenrodt, A., and Lang, J . M. (1985) Isoprinosine and Imuthiol,
two potentially active compounds in patients with aids related
complex symptoms. Cancer Res. 45, 4671s-4673s.
2
gavage. Similarly, decreased formation of TTCA may
have resulted in the present investigation from a more
(
9) Neveu, P. J ., and Perdoux, D. (1986) Evaluation of the mecha-
nisms involved in sodium diethyl dithiocarbamate-induced im-
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D-glucamine dithiocarbamate. Int. Arch. Allergy Appl. Immunol.
rapid uptake of CS
2
following ip injection as compared
to po administration.
There has been previous documentation for the pres-
ence of TTCA in the urine of humans and rats exposed
8
0, 164-167.
(10) Eneanya, D. I., Bianchine, J . R., Duran, D. O., and Andresen, B.
D. (1981) The actions and metabolic fate of disulfiram. Annu. Rev.
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(12) Mokri, B., Ohnishi, A., and Dyck, P. J . (1981) Disulfiram neur-
to CS
2
. In the present study, we have used TTCA to
from dithiocar-
further establish in vivo release of CS
2
(
bamates as a potential mechanism of toxicity. We have
demonstrated TTCA in the urine of rats challenged with
DEDC, DS, and NMDC and have also shown that the
CS released in vivo by DEDC is incorporated into TTCA
2
at the thiocarbonyl carbon. Levels of TTCA produced by
these compounds followed the pattern predicted from the
opathy. Neurology 31, 730-735.
(
13) Komulainen, H., and Savolainen, K. (1985) Effect of dithiocar-
3
bamate fungicides and thiurams on H-haloperidol binding in rat
brain. Arch. Toxicol. Suppl. 8, 77-79.
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a review (1971-1988) and report of a case. Alcohol Alcohol. 24,
molar content of CS
promoted decomposition to parent amine and CS
2
and the relative rates of acid-
ob-
4
29-437.
2
(
15) Ansbacher, L. E., Bosch, E. P., and Cancilla, P. A. (1982)
Disulfiram neuropathy: A neurofilamentous distal axonopathy.
Neurology 32, 424-428.
served in vitro. It is interesting to note that oral exposure
to dithiocarbamates resulted in greater levels of TTCA
relative to parenteral administration and that DS and
DEDC produced levels of TTCA that were greater than
(16) Bilbao, J . M., Briggs, S. J ., and Gray, T. A. (1984) Filamentous
axonopathy in disulfiram neuropathy. Ultrastruct. Pathol. 7, 295-
3
00.
2
and equivalent to CS , respectively. Although somewhat
(
17) Kane, J ., and Francis J . (1970) Carbon disulfide intoxication from
useful for evaluating acute exposures, the rapid elimina-
tion of TTCA following cessation of exposure suggests
that a biomarker demonstrating a cumulative dose
response, as observed for spectrin cross-linking, may be
more suitable for monitoring chronic exposures as occur
in DS therapy. The differences observed here in TTCA
levels resulting from route of exposure may reflect
differences in the bioavailability of CS that are relevant
2
when considering routes of administration for dithiocar-
bamate-based medical agents.
overdosage of disulfiram. Am. J . Psychiatry 127, 690-694.
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of monoalkyl dithiocarbamates. Anal. Chem. 42, 647-651.
(
19) Miller, D. M., and Latimer, R. A. (1962) The kinetics of the
decomposition and synthesis of some dithiocarbamates. Can. J .
Chem. 40, 246-255.
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T. (1975) Decomposition of dithiocarbamates. IX. The effect of
carbon disulfide on the decomposition of N-monosubstituted
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Graham, D. G. (1995) Carbon disulfide mediated protein cross-
linking by N,N-diethyldithiocarbamate. Chem. Res. Toxicol. 8,
9
6-102.
Ack n ow led gm en t. We gratefully acknowledge the
Duke Magnetic Resonance Center for use of the facilities,
and D.J .J . gratefully acknowledges funding support from
an NSF predoctoral fellowship (1993-94), National In-
stitute of Environmental Health Sciences, NIH Grants
ES07031 (1994-95), and ES07028 (1995-present). This
publication was made possible by Grant ES06387 from
the National Institute of Environmental Health Sciences,
NIH.
(22) Valentine, W. M., Graham, D. G., and Anthony, D. C. (1993)
Covalent cross-linking of erythrocyte spectrin by carbon disulfide
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D. C. (1992) Covalent cross-linking of proteins by carbon disulfide.
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(
(
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Environ. Health 36, 289-297.
(
25) Bus, J . S. (1985) The relationship of carbon disulfide metabolism
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American Chemical Society Division of Chemical Toxicology (probation-
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