(Received for publication, June 22, 1995)
From the
Dithiocarbamates are metal-chelating compounds that can exert either pro-oxidant or antioxidant effects in different situations. They have recently been found to potently inhibit apoptotic cell death, an activity attributed to their antioxidant action. However, when thymocytes were exposed to pyrrolidine dithiocarbamate, an oxidation of the glutathione pool occurred within 90 min. Longer incubation resulted in cell shrinkage, chromatin fragmentation, glutathione depletion, and eventual cell lysis, which is typical of apoptosis in these cells. These changes were inhibited by inclusion of non-permeable metal chelators in the incubation medium, suggesting that pyrrolidine dithiocarbamate exerts its toxic effect by transporting a redox-active metal into the cell. This was directly confirmed when sustained 8-fold elevations of intracellular copper were detected after addition of pyrrolidine dithiocarbamate. In agreement with this, supplementation of the incubation medium with submicromolar concentrations of copper significantly potentiated pyrrolidine dithiocarbamate toxicity. We conclude that pyrrolidine dithiocarbamate exerts a powerful pro-oxidant effect on thymocytes due to its ability to transport external redox-active copper into cells. The resulting increase in glutathione disulfide may also explain the temporary anti-apoptotic activity of this compound described in other systems.
Dithiocarbamates (DCs) ()are known to exert
pro-oxidant and antioxidant effects in both cell-free and biological
systems. Their biological effects include widespread use as
agricultural insecticides, herbicides, and fungicides with an estimated
annual global consumption of 25,000-35,000 metric
tons(1) . They have been used clinically in the treatment of
various pathogenic fungi and bacteria (2) and recently even in
the experimental therapy of AIDS(3) . Their diverse functions
also include use in aversion therapy against chronic alcoholism
(disulfiram/Antabus(TM)) and as an antidote against nickel and
copper poisoning(4) . Many of the biological effects of DCs are
based on their metal-chelating properties, and they have also been used
for many years in analytical methods for determination of heavy metals,
especially in organic samples due to their relatively lipophilic
nature(2) . In addition to binding metals, the free thiol
groups of DCs can also react with sulfhydryl groups on other molecules.
DCs have thus been reported to inhibit enzymes by covalent interaction
to free protein thiols (2, 5) as well as to oxidize
glutathione through a glutathione peroxidase-like
activity(6, 7) . DCs may also interfere with cellular
detoxication mechanisms as they are reported to suppress hepatic
microsomal drug metabolism (8) and to inhibit glutathione S-transferases(9) . In addition, the
diethyldithiocarbamate (DDC) derivative has been found to inhibit
copper/zinc superoxide dismutase activity by withdrawal of essential
metal from the enzyme (10, 11) and to deplete
intracellular glutathione in a non-superoxide dismutase-dependent
manner(12, 13) .
A problem in evaluating the
biochemical action of many DCs is that they are unstable, decomposing
via two different pathways to form biologically active metabolites.
Under acidic conditions, the decomposition of DCs to their
corresponding amine and CS is favored(14) .
CS
mediates protein cross-linking and is proposed to be an
important molecule behind DDC-induced toxicity(15) . Second,
DCs can be metabolized by cytochrome P-450 to a S-methyl
sulfoxide derivative, which in turn is a potent inhibitor of enzymes
such as aldehyde dehydrogenase. Disulfiram is believed to exert its
effects after this type of P-450-dependent
metabolism(16, 17) . The stability of DCs can be
increased by modifying the aliphatic substitutions on the nitrogen
atom. An example is pyrrolidine dithiocarbamate (PDTC), a much more
stable analog of DDC(14) .
DCs have recently found use in
cell and molecular biology as antioxidants. Cell-free antioxidant
effects include inhibition of lipid peroxidation in both liposomes and
liver microsomes(18, 19, 20) , while it has
been widely assumed that similar effects occur when DCs are applied to
intact cells. For example, PDTC potently inhibits oxidative activation
of the transcription factor
NFB(21, 22, 23) , while others report
that the same compound inhibits apoptosis in thymocytes(24) ,
leukemic cells(25) , and in L929 fibroblasts(26) .
Reactive oxygen species have been proposed to be common mediators of
apoptosis in many different cell systems(27) , while
antioxidants are often observed to exert an inhibitory effect on this
type of cell death(24, 28) . The widespread use of DCs
in medicine and industry and its cytotoxic action in many cells,
combined with reports of both pro-oxidant and antioxidant effects, led
us to investigate whether a general antioxidant activity was indeed
responsible for its ability to interfere with the apoptotic process. We
now report that PDTC exerts a toxic pro-oxidant effect in thymocytes,
inducing apoptotic cell shrinkage and chromatin fragmentation prior to
cell lysis. This toxicity is dependent on the ability of the compound
to transport external copper into the cells and thereby generate an
intracellular oxidative stress.
PDTC has been described as a potent inhibitor of thymocyte
apoptosis(24, 25) , and in confirmation of this we
have found that it inhibits DNA fragmentation in thymocytes exposed to
etoposide, glucocorticoids, or thapsigargin (IC
approximately 10-20 µM PDTC in each case; data not
shown). As DCs are reported to protect against oxygen toxicity in
vivo(35, 36) and to possess antioxidant
properties in vitro(18, 19, 20) ,
and antioxidants in general are known to delay apoptosis in many model
systems, it has been assumed that the metal-chelating and/or
radical-scavenging properties of PDTC mediate its anti-apoptotic
activity.
We have previously shown that a decrease of both intracellular GSH and reduced protein thiol is an early event in the apoptotic process of thymocytes that can be prevented by incubating the cells with various antioxidants(28) . However, when the GSH levels of thymocytes induced to undergo apoptosis in the presence of PDTC were measured, the dithiocarbamate was observed to inhibit DNA fragmentation without reversing GSH depletion (Fig. 1A). PDTC treatment alone was actually seen to oxidize the intracellular glutathione pool; GSSG content was elevated 1.5, 3, and 6 h after treatment, while GSH levels were progressively reduced by 6 h and thereafter (Fig. 1B). These results demonstrate that low concentrations of PDTC exert a pro-oxidant effect on intracellular GSH/GSSG redox in thymocytes, and therefore a general antioxidant effect cannot explain the anti-apoptotic activity of this compound.
Figure 1:
PDTC inhibition of etoposide-induced
DNA fragmentation correlates with an oxidation and depletion of
intracellular GSH. Thymocytes were incubated at 37 °C, and
intracellular levels of GSH were determined at various time points. A, cells were untreated () or cultured with 25
µM etoposide (
) or 25 µM etoposide plus
20 µM PDTC (
). DNA fragmentation was determined in
the same samples using diphenylamine. All results are mean ±
S.D. (n = 3). B, average GSH (
) and GSSG
(
) levels (standardized against the levels in untreated cells) of
cells treated with 20 µM PDTC are shown after different
times of incubation.
The observation that PDTC exerts a pro-oxidant activity on the glutathione pool of thymocytes suggested that it could be toxic during longer incubations. Indeed, cell membrane permeability (measured with trypan blue) was markedly reduced when thymocytes were incubated overnight with different doses of PDTC (Fig. 2A). Even 4 µM PDTC was significantly toxic to the cells, and at 40 µM PDTC the cell viability fell below 50%. Lysis of the cells was accompanied by an increase in low molecular weight DNA fragmentation (Fig. 2B), although DNA damage was already evident at 1 µM PDTC before any loss in cell viability could be detected. To further investigate the mechanism of this cell death, a time-course study of the effect of PDTC on chromatin degradation and cell volume was undertaken (Fig. 3, A and B). 20 µM PDTC was found to give a time-dependent induction of DNA fragmentation, first evident after 6 h and increasing thereafter, while significant reductions in cell volume occurred with similar kinetics. When pulsed field gel electrophoresis on DNA samples from the same experiment was performed, 50-kb DNA fragments were present by 6 h (Fig. 3C). Formation of 200- and 50-kb DNA fragments was concentration dependent, increasing from 1 to 40 µM PDTC (8-h incubation, Fig. 3D). As cell membrane permeability was not affected by PDTC at this time (Fig. 2A), these data indicate that the DC induces an apoptotic response (characterized by chromatin fragmentation and cell shrinkage) prior to any secondary lysis of the cells. In parallel experiments, DDC (40 µM) also gave a significant enhancement of thymocyte DNA fragmentation at 6 h (data not shown), indicating that these effects are specific for the shared dithiocarbamate structure.
Figure 2:
PDTC induces a concentration-dependent
cell lysis and DNA fragmentation. Thymocytes were incubated at 37
°C for 8 h () or 24 h (
) with different doses of PDTC;
then, cell viability was assayed by the trypan blue exclusion method
(2-300 cells counted per sample) (A), and the amount of
fragmented DNA was determined using the diphenylamine assay (mean
± S.D., n = 3) (B).
Figure 3:
Time- and dose-dependent induction of
thymocyte apoptosis by PDTC. Thymocytes were incubated at 37 °C for
different times as indicated, and then the amount of fragmented DNA was
determined (mean ± S.D., n = 3) (A) or
the mean cell volume was estimated using a Coulter counter
(approximately 5000 cells counted per sample) (B). Cells were
untreated () or cultured with 20 µM PDTC (
). C, samples were also processed for field-inversion gel
electrophoresis analysis (see ``Experimental Procedures'').
Gels were stained with 0.5 µg
ml
ethidium bromide for 1 h and visualized with a UV lamp. DNA
equivalent to approximately 0.5
10
cells was loaded
in each lane, and the sizes were estimated with
Pulse-marker
. D, samples taken from the
experiment described in Fig. 2were analyzed by field-inversion
gel electrophoresis as described above. Cells were treated with PDTC
(shown in lanes 1-6 in the order of 0, 1, 4, 10, 20, 40
µM PDTC) for 8 h at 37 °C.
By virtue of its strong metal-chelating properties and high membrane permeability(2) , DDC has been reported to transport metal ions across cell membranes(37) . The ability of a series of phenanthroline metal chelators to interfere with PDTC-induced apoptosis of thymocytes was therefore tested. Both the membrane-permeable copper-specific chelator neocuproine and its non-permeable analog BCPS were observed to inhibit the DNA fragmentation that occurred after thymocytes were exposed to PDTC (Table 2). BCPS protection was dose dependent (data not shown) and was gradually overcome when PDTC concentrations were raised above 10 µM (Fig. 4). BPS was also active as an inhibitor of PDTC toxicity (Table 2). As this non-permeable chelator is thought to be an iron-specific reagent, these experiments cannot identify the metal ion involved, while the lack of effect observed with the nonchelating 1,7-phenanthroline demonstrates that it is the metal-chelating properties of these compounds that interfere with PDTC toxicity (Table 2). Both BCPS and BPS were completely inactive as inhibitors of etoposide-induced DNA fragmentation, while as reported previously, neocuproine exerted a weak protective effect ( Table 2and (24) ).
Figure 4:
BCPS inhibits cell lysis and DNA
fragmentation induced by PDTC. Thymocytes were incubated at 37 °C
overnight with different doses of PDTC, when cell viability was
determined as described in Fig. 2(A) and DNA
fragmentation were assayed (B) in cells treated with ()
or without 50 µM BCPS (
). The data are pooled from
three separate experiments and are expressed as mean ±
S.D.
The above results suggest that a PDTC-dependent transport of external redox-active metal(s) into thymocytes was responsible for its toxicity. GFAAS was therefore employed to identify the metal involved (Table 3). The intracellular copper content of thymocytes was raised 8-fold after a 1.5-h incubation with 10 µM PDTC. The copper content of PDTC-treated thymocytes remained high at all later time points but was almost completely abrogated if the cells were co-incubated with 50 µM BCPS. In this experiment, untreated cells showed a transient 2-fold increase of copper at 1.5 h, but levels at time points thereafter returned to the base line. No increase in intracellular iron was detected after any of the treatments (Table 3). To verify that this increase of intracellular copper mediated the oxidation observed during PDTC exposure, the intracellular levels of GSH and GSSG were determined in a similar experiment. GSSG levels were significantly elevated 1.5 h after adding PDTC, while co-incubation of the cells with the external copper chelator BCPS blocked this oxidation of the glutathione pool (Fig. 5).
Figure 5:
BCPS
inhibits PDTC-induced formation of GSSG. Thymocytes were incubated for
various times at 37 °C, and samples were then taken for
determination of intracellular GSSG. Cells were untreated (),
incubated with 10 µM PDTC (
), or co-cultured with 10
µM PDTC and 50 µM BCPS (
). Results
show mean ± S.D. from triplicate
samples.
These experiments indicate that
PDTC transports redox-active external copper across the thymocyte cell
membrane, thereby generating an oxidative stress within the cell that
in turn induces apoptosis. Copper was not added to the media in any of
these experiments, and therefore trace amounts of the metal (probably
in the fetal calf serum supplement) are apparently sufficient for this
process to occur. This suggested that supplementation of the media with
external copper would potentiate PDTC toxicity. Consistent with this,
in the presence of suboptimal 0.1 or 1 µM PDTC, addition
of CuSO to the culture media substantially increased DNA
fragmentation and cell lysis. When the CuSO
concentration
was raised above 0.4 µM, addition of 1 µM
PDTC promoted necrotic rather than apoptotic cell death, reflected in
reduced DNA fragmentation, and increased cell lysis (Fig. 6).
Supplementation of the media with up to 1.6 µM CuSO
alone was not toxic to thymocytes in overnight incubation (Fig. 6).
Figure 6:
Copper sulfate potentiates PDTC-induced
cell lysis and DNA fragmentation. Thymocytes were incubated overnight
at 37 °C with various doses of CuSO and 0 µM PDTC
), 0.1 µM PDTC (
), or 1 µM PDTC (
). A, DNA fragmentation was assayed using
diphenylamine; B, cell viability was determined with the
trypan blue exclusion method (see Fig. 2legend for
details).
Our results demonstrate that transport of extracellular
copper from the media into cells is required for PDTC to induce
apoptosis in thymocytes. The calf sera supplement is the most probable
source of external copper in our experiments, as sera normally contain
from 0.3-0.6 ppm of copper in mice (38) to 1 ppm of
copper in humans(39) . Elevated serum copper levels correlates
with the progress of some lymphocyte-associated diseases such as
AIDS(39) , lymphomas(40) , and also in age-related
immune deficiency(38) . We show here that increased copper
levels in thymocytes generates an oxidative stress as measured by an
increase in intracellular GSSG. The oxidative stress in turn is
cytotoxic, inducing apoptosis at lower PDTC concentrations and necrotic
lysis when the stress is enhanced (Fig. 2). Other oxidants
including menadione and diamide have been reported to cause a similar
dose-dependent cytotoxicity, where low amounts of oxidant induce
apoptosis, whereas higher concentrations give rise to necrosis (41, 42) . Copper is known to redox cycle and thereby
generate reactive oxygen species such as hydroxyl radical
(OH)(43) . Interestingly, the chelating agent
1,10-phenanthroline has been shown to enhance copper-dependent
production of OH
(44) . As PDTC has a log
of 10.9 for Cu
(45) and has
not been reported to bind Cu
, it is probably the
former that PDTC transports into cells. This
Cu
PDTC complex may be reduced by endogenous
reductants (GSH or ascorbic acid) and then redox cycle in the presence
of oxygen. The resulting formation of reactive oxygen species would
oxidize glutathione, probably in a very efficient way as DDC is
reported to have a glutathione peroxidase-like
activity(6, 7) . Ultimately, the GSH pool of the cells
would be depleted, consistent with observations in other
systems(12, 13) .
Since DDC has been reported to inhibit copper/zinc superoxide dismutase (10, 11) , this provides another potential mechanism by which DCs can mediate an oxidative stress. However, this is unlikely to be the case in this study as millimolar concentrations of DDC are required to inhibit superoxide dismutase(13) . The fact that we detected an increase in intracellular level of copper after treatment with PDTC (Table 3) also strongly suggests that inhibition of superoxide dismutase is not relevant because such inhibition would be reduced, not potentiated, by addition of copper. The observation of Mohindru et al.(46) that extracellular copper potentiates a DDC-induced arrest of cell proliferation further supports our findings that a direct copper-dependent oxidative stress is responsible for the cytotoxic effects of PDTC.
Bioactivation of DDC to a S-methyl sulfoxide derivative is known to be catalyzed by
cytochrome P-450(16, 17) . In an attempt to determine
whether the catabolism of PDTC was required to mediate its toxic
effect, thymocytes were co-incubated with PDTC and metyrapone (a
nonspecific cytochrome P-450 inhibitor). However, no effect on PDTC
toxicity was seen (data not shown), indicating that cytochrome
P-450-dependent bioactivation is not involved in the toxic effect of
PDTC in our system. DDC can spontaneously decompose to diethylamine and
CS, where CS
has well documented
toxicity(47) . This type of reaction does not seem to be
crucial in our system since the transport of copper is necessary for
PDTC to mediate its toxic action, although the decomposition of PDTC
inside cells may facilitate the intracellular release of chelated
copper and thereby enhance the potential for oxidative damage to occur.
As mentioned above, PDTC has a completely different biological
effect in a shorter time scale as it (a) prevents both
thymocyte and leukemic cell apoptosis induced by different agents (24, 25) and (b) inhibits the activation of
NFB and enhances the DNA binding of AP-1. Since these
transcription factors are redox-regulated, and many apoptotic cells
experience an oxidative stress, both of these effects have been assumed
to be due to a general metal-chelating and/or radical-scavenging
property of
PDTC(21, 22, 23, 24, 26) .
However, as PDTC is active at micromolar concentrations in the presence
of millimolar intracellular GSH, it is unlikely that it exerts a
significant effect by directly scavenging radicals. We find that it
actually oxidizes GSH to GSSG within 1.5 h of treatment, i.e. in the same time period when it exerts an effect on transcription
factor activity and apoptosis. Correlation between an increase of GSSG
and inhibition of NF
B activation by PDTC has been observed
before(48) , which makes it tempting to speculate that a GSSG
increase mediates many of the inhibitory/regulatory effects of this
compound. Preliminary experiments show that we can block the inhibitory
action of PDTC on etoposide apoptosis by adding the external copper
chelator BCPS, indirectly supporting the hypothesis that GSSG is
involved in its anti-apoptotic activity. GSSG is known to be an
important mediator of protein function, for example in the regulation
of NF
B activation and DNA binding(49) . It is therefore
possible that the pro-oxidant effect of PDTC is responsible both for
the initial prevention and subsequent induction of apoptosis. Similar
phenomena involving oxidative stress have been observed before; for
example, increasing the concentration of a redox-cycling quinone shifts
the cellular response from proliferation to apoptosis(41) . We
are currently investigating the possibility that PDTC regulatory
effects on apoptosis involves GSSG modulation of crucial
enzymes/proteins in the machinery of apoptosis.