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(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) ( 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 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
NF
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 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 (
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 (
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 (
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 (
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 (
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
Figure 6:
Copper sulfate potentiates PDTC-induced
cell lysis and DNA fragmentation. Thymocytes were incubated overnight
at 37 °C with various doses of CuSO
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 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 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
NF
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26202-26208
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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) .
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) .
B(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.
Materials
PDTC, DDC,
2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonic acid
(bathocuproine disulfonic acid or BCPS),
4,7-diphenyl-1,10-phenanthroline disulfonic acid (bathophenanthroline
disulfonic acid or BPS), and 1,7-phenanthroline were obtained from
Aldrich. Molecular biology grade of the sodium salt and free acid of
Hepes (buffer in the sample preparation for GFAAS), etoposide,
6-
-methylprednisolone, 2,9-dimethyl-1,10-phenanthroline
(neocuproine), diphenylamine, dansyl chloride, N-lauroyl
sarcosine, and Pulse marker (0.1-200 kb, 225-1000
kb) were bought from Sigma. Low melting point agarose (Sea Plaque
GTG-agarose) and agarose (Sea Kem GTG-agarose) were purchased from FMC
Bioproducts, and Proteinase K came from Boehringer Mannheim. Reagents
for cell culture including fetal calf serum, RPMI 1640, and gentamicin
were from Life Technologies, Inc. Determinations of protein levels were
made with the bicinchoninic acid (BCA) reagent kit from Pierce.
Thymocyte Preparation and
Culture
5-8-week-old (70-100 g) male
Sprague-Dawley rats from B& Universal (Sollentuna, Sweden) were
sacrificed in a CO ventilated box, and the thymic glands
were excised. Preparation of thymocytes for culture was made as
described(29) . For experiments, cells were incubated at a
density of 5 
10
ml in
RPMI 1640 supplemented with either 2% (incubations
6 h) or 5%
fetal calf serum and 10 µg ml gentamicin
in an atmosphere containing 5% CO
. Cell viability was
determined before each experiment by trypan blue exclusion and was
always greater than 95%.Quantitation of Apoptosis
DNA
fragmentation was quantitated with the diphenylamine assay as described
earlier(30) . The formation of high molecular weight DNA
fragments was assayed by field-inversion gel electrophoresis according
to Zhivotovsky et al.(31) but using a vertical
electrophoresis system and modified electrophoretic running
conditions(32) . The distribution of cell volumes in a sample
of 5 10
thymocytes was measured in a Z1 Coulter
counter (Coulter Electronics Ltd., Luton, UK), and the results were
expressed as the mean cell volume in each sample.GSH and GSSG Determinations
Samples
consisting of 2 10
thymocytes were harvested and
washed twice in ice-cold phosphate-buffered saline (pH 7.2). An aliquot
was removed for protein determination, and remaining proteins were then
precipitated with 1 M perchloric acid in 0.2 M boric
acid. Samples were centrifuged, and the supernatants were derivatized
with 6 mM iodoacetic acid at pH 9.0 for 30 min. Dansyl
chloride was added to give a concentration of 2 mM, and
samples were left for 24 h to form S-carboxymethyl-N-dansyl-GSH and N,N`-bis-dansyl-GSSG. Unreacted dansyl
chloride was extracted by adding chloroform to the samples. The GSH and
GSSG adducts were then separated by HPLC essentially as described by
Reed et al.(33) for the corresponding dinitrophenyl
adducts and quantified relative to standards using a fluorescence
detector (excitation wavelength, 335 nm, emission wavelength, 515 nm).Intracellular Copper and Iron
Determination
Precautions were made to avoid contamination
by copper and iron according to Zhang et al.(34) , and
the Hepes buffer and acid used were found to have negligible contents
of copper and iron. Samples consisting of 5 10
cells were washed three times in 15 mM Hepes (pH calibrated by
using only sodium salt and free acid) in 0.9% (w/v) NaCl (fresh bottle
pro analysi), pH 7.3, to minimize external contaminations of copper and
iron. The cells were suspended in the Hepes buffer, and aliquots for
protein determination were removed. 0.3 ml of the cell suspension was
solubilized by the addition of 0.9 ml of 0.1 M HNO (pro analysi), heated at 60 °C for 3-4 h, and then left
overnight at 4 °C. The final slightly turbid solution was used for
the measurements. Copper and iron concentrations were determined by
GFAAS, using an instrument (Perkin-Elmer 5000 Zeeman) equipped with an
electrothermal atomization unit (HGA-500) and an automatic sample
injector (AS-40). The analysis was carried out essentially as given in (34) with a modified atomization program (Table 1).
Stabilized temperature platform furnace technique was utilized;
however, for iron an internal gas flow (Table 1) was used due to
relatively high concentrations. The samples were quantified using
external standards diluted to the same final acid (75 mM HNO) and buffer concentrations as in the samples.
Standards were bought as stock standard solutions from BDH (UK). The
sample matrix effect was controlled by adding standards to the samples
and was found to be insignificant. Results were standardized according
to total cellular protein.
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.
) 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.
) 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).
) 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.
)
or without 50 µM BCPS (
). The data are pooled from
three separate experiments and are expressed as mean ±
S.D.
),
incubated with 10 µM PDTC (), or co-cultured with 10
µM PDTC and 50 µM BCPS (
). Results
show mean ± S.D. from triplicate
samples.
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).
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).
)(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) ., 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. B 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 NFB 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 NFB 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.
)B, nuclear transcription factor B; HPLC, high pressure
liquid chromatography; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;
kb, kilobase(s); GSSG, glutathione disulfide.
Kerstin Ström is acknowledged for
assistance with the setup of the HPLC system, Bo Nilsson for help with
the GFAAS measurements, and Elisabeth Wakeman for secretarial
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 A. Iseki, F. Kambe, K. Okumura, T. Hayakawa, and H. Seo Regulation of Thyroid Follicular Cell Function by Intracellular Redox-Active Copper Endocrinology, December 1, 2000; 141(12): 4373 - 4382. [Abstract] [Full Text] [PDF]
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 W. Erl, C. Weber, and G. K. Hansson Pyrrolidine dithiocarbamate-induced apoptosis depends on cell type, density, and the presence of Cu2+ and Zn2+ Am J Physiol Cell Physiol, June 1, 2000; 278(6): C1116 - C1125. [Abstract] [Full Text] [PDF]
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H. Ishiyama, N. C. Hoglen, and I. G. Sipes Diethyldithiocarbamate Enhances Production of Nitric Oxide and TNF-{alpha} by Lipopolysaccharide-Stimulated Rat Kupffer Cells Toxicol. Sci., May 1, 2000; 55(1): 206 - 214. [Abstract] [Full Text] [PDF]
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 D. N. Muller, R. Dechend, E. M. A. Mervaala, J.-K. Park, F. Schmidt, A. Fiebeler, J. Theuer, V. Breu, D. Ganten, H. Haller, et al. NF-{kappa}B Inhibition Ameliorates Angiotensin II-Induced Inflammatory Damage in Rats Hypertension, January 1, 2000; 35(1): 193 - 201. [Abstract] [Full Text] [PDF]
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 K. Aoshiba, S. Yasui, K. Nishimura, and A. Nagai Thiol Depletion Induces Apoptosis in Cultured Lung Fibroblasts Am. J. Respir. Cell Mol. Biol., July 1, 1999; 21(1): 54 - 64. [Abstract] [Full Text]
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 M Schweizer and E Peterhans Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis J. Gen. Virol., May 1, 1999; 80(5): 1147 - 1155. [Abstract]
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 F. Salvemini, A. Franze, A. Iervolino, S. Filosa, S. Salzano, and M. V. Ursini Enhanced Glutathione Levels and Oxidoresistance Mediated by Increased Glucose-6-phosphate Dehydrogenase Expression J. Biol. Chem., January 29, 1999; 274(5): 2750 - 2757. [Abstract] [Full Text] [PDF]
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 C. L. Hartsfield, J. Alam, and A. M. K. Choi Transcriptional regulation of the heme oxygenase 1 gene by pyrrolidine dithiocarbamate FASEB J, December 1, 1998; 12(15): 1675 - 1682. [Abstract] [Full Text]
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B. Friedrichs, C. Muller, and R. Brigelius-Flohe Inhibition of Tumor Necrosis Factor-{alpha}– and Interleukin-1–Induced Endothelial E-Selectin Expression by Thiol-Modifying Agents Arterioscler. Thromb. Vasc. Biol., December 1, 1998; 18(12): 1829 - 1837. [Abstract] [Full Text] [PDF]
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H.-H. Wu and J. Momand Pyrrolidine Dithiocarbamate Prevents p53 Activation and Promotes p53 Cysteine Residue Oxidation J. Biol. Chem., July 24, 1998; 273(30): 18898 - 18905. [Abstract] [Full Text] [PDF]
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 Q. Chen and A. I. Cederbaum Cytotoxicity and Apoptosis Produced by Cytochrome P450 2E1 in Hep G2 Cells Mol. Pharmacol., April 1, 1998; 53(4): 638 - 648. [Abstract] [Full Text]
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A. G. Bowie, P. N. Moynagh, and L. A. J. O'Neill Lipid Peroxidation Is Involved in the Activation of NF-kappa B by Tumor Necrosis Factor but Not Interleukin-1 in the Human Endothelial Cell Line ECV304. LACK OF INVOLVEMENT OF H2O2 IN NF-kappa B ACTIVATION BY EITHER CYTOKINE IN BOTH PRIMARY AND TRANSFORMED ENDOTHELIAL CELLS J. Biol. Chem., October 10, 1997; 272(41): 25941 - 25950. [Abstract] [Full Text] [PDF]
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G. T. Gobbel, T. Y.-Y. Chan, and P. H. Chan Nitric Oxide- and Superoxide-Mediated Toxicity in Cerebral Endothelial Cells J. Pharmacol. Exp. Ther., September 1, 1997; 282(3): 1600 - 1607. [Abstract] [Full Text]
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Q. Chen, M. Galleano, and A. I. Cederbaum Cytotoxicity and Apoptosis Produced by Arachidonic Acid in Hep G2 Cells Overexpressing Human Cytochrome P4502E1 J. Biol. Chem., June 6, 1997; 272(23): 14532 - 14541. [Abstract] [Full Text] [PDF]
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C. M. Krejsa, S. G. Nadler, J. M. Esselstyn, T. J. Kavanagh, J. A. Ledbetter, and G. L. Schieven Role of Oxidative Stress in the Action of Vanadium Phosphotyrosine Phosphatase Inhibitors. REDOX INDEPENDENT ACTIVATION OF NF-kappa B J. Biol. Chem., April 25, 1997; 272(17): 11541 - 11549. [Abstract] [Full Text] [PDF]
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D. J.v. d. Dobbelsteen, C. S. I. Nobel, J. Schlegel, I. A. Cotgreave, S. Orrenius, and A. F.G. Slater Rapid and Specific Efflux of Reduced Glutathione during Apoptosis Induced by Anti-Fas/APO-1 Antibody J. Biol. Chem., June 28, 1996; 271(26): 15420 - 15427. [Abstract] [Full Text] [PDF]
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R. Pinkus, L. M. Weiner, and V. Daniel Role of Oxidants and Antioxidants in the Induction of AP-1, NF-kappa B, and Glutathione S-Transferase Gene Expression J. Biol. Chem., June 7, 1996; 271(23): 13422 - 13429. [Abstract] [Full Text] [PDF]
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J. J. E. Haddad, R. E. Olver, and S. C. Land Antioxidant/Pro-oxidant Equilibrium Regulates HIF-1alpha and NF-kappa B Redox Sensitivity. EVIDENCE FOR INHIBITION BY GLUTATHIONE OXIDATION IN ALVEOLAR EPITHELIAL CELLS J. Biol. Chem., July 7, 2000; 275(28): 21130 - 21139. [Abstract] [Full Text] [PDF]
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