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Oxidativestressinducesareversiblefluxofcysteine from
tissues tobloodinvivointhe rat
Daniela Giustarini
1
, Isabella Dalle-Donne
2
, Aldo Milzani
2
and Ranieri Rossi
1
1 Department of Evolutionary Biology, Laboratory of Pharmacology and Toxicology, University of Siena, Italy
2 Department of Biology, University of Milan, Italy
Introduction
Cysteine (Cys) and glutathione (GSH) are the most
abundant low-molecular-mass thiols (LMM-SH), with
GSH predominating intracellularly and cysteine
predominating in extracellular fluids [1,2]. These two
compounds are metabolically inter-related, and GSH,
in particular, determines the redox state and represents
a defense against damage mediated by reactive oxygen
species (ROS) or reactive nitrogen species.
Low GSH concentrations and a ratio of high gluta-
thione disulfide (GSSG) to GSH have been measured
in red blood cells (RBCs) of patients with various
diseases, including cancer, coronary heart surgery,
pre-eclampsia, and genitourinary, gastrointestinal, car-
diovascular and musculoskeletal diseases [3–9]. Extra-
cellular fluids such as plasma are characterized by a
lower thiol : disulfide ratio in comparison tothe intra-
cellular environment, and many pathophysiological
conditions has been shown to markedly influence both
thiol and disulfide concentrations [1,10–13].
GSH is synthesized intracellularly from its constituent
amino acids, cysteine, glutamate and glycine, by a two-
step reaction catalyzed by the enzymes c-glutamylcyste-
ine synthetase and GSH synthetase. The supply of Cys
is directly related tothe rate of GSH synthesis, because
the cellular cysteine concentration is a key determinant
in regulating the kinetics ofthe first reaction (i.e. the
formation of c-glutamylcysteine), which represents the
rate-limiting step in GSH synthesis [14]. Cysteine is rap-
idly auto-oxidized to cystine (CySS) inthe extracellular
fluids; CySS may have a considerable physiological role
as a source of Cys, as, once it has entered a different cell,
it can be reduced again by GSH [15].
Keywords
cysteine; diamide; glutathione; oxidative
stress; thiols
Correspondence
R. Rossi, Department of Evolutionary
Biology, Laboratory of Pharmacology and
Toxicology, University of Siena, via A. Moro
4, I-53100 Siena, Italy
Fax: +39 577 234476
Tel: +39 577 234198
E-mail: ranieri@unisi.it
(Received 11 May 2009, revised 30 June
2009, accepted 3 July 2009)
doi:10.1111/j.1742-4658.2009.07197.x
Glutathione (GSH) plays a key role in defense against oxidative stress. The
availability of GSH is ensured intissues by systems devoted to its mainte-
nance inthe reduced state and by thefluxof GSH and cysteine between
sites of biosynthesis and sites of utilization. Little is known about the effect
of oxidativestress on the distribution of low-molecular-mass thiols and
their exchange rate between tissues. In this study, we found that a slow
infusion of diamide (a specific thiol-oxidizing compound) evoked a dra-
matic increase inbloodcysteinein rats. Our data suggest that inter-organ
exchange ofcysteine occurs, that cysteine derives from both glutathione via
c-glutamyl transpeptidase and methionine via homocysteine and the trans-
sulfuration pathway, and that these pathways are considerably influenced
by oxidative stress.
Abbreviations
CySS, cystine; CysGly, cysteinylglycine; CySSGly, cystinylglycine; GSH, glutathione; GSSG, glutathione disulfide; GSSP, mixed disulfides
between protein sulfhydryl groups and glutathione; c-GT, c-glutamyl transpeptidase; Hcys, homocysteine; HcySS, homocystine; LMM-SH,
low-molecular-mass thiols; mBrB, monobromobimane; NEM, N-ethylmaleimide; RBC, red blood cell; ROS, reactive oxygen species; RSSP,
mixed disulfides between protein sulfhydryl groups and a low-molecular-mass thiol; TSP, trans-sulfuration pathway.
4946 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS
A continuous fluxof GSH and Cys exists between
various tissues, and diet, starvation and pathologies
that affect various organs may influence this phenome-
non [2,16,17]. Many pathological conditions are known
or thought to elicit oxidative stress, and it is recog-
nized that this may have a role inthe progression of
the disease (for review, see [18]). Usually, the occur-
rence ofoxidativestress is evaluated by measuring sev-
eral biomarkers, but little is known about the influence
of oxidativestress on cysteine and glutathione
exchange among tissues. Indeed, the factors that main-
tain the redox homeostasis of plasma thiols as well as
the mechanisms that regulate the exchange of LMM-
SH among cells need to be clarified.
Here, we have analyzed the effect ofthe administra-
tion of azodicarboxylic acid bis-dimethylamide (dia-
mide), a thiol-specific oxidizing substance, on the
blood and tissue distribution of thiols and disulfides in
rats. Diamide is a mild oxidizing compound that easily
penetrates cell membranes and reacts quickly and spe-
cifically with intracellular thiols, both LMM-SH and
protein sulfhydryls [19,20], without producing free rad-
icals or other ROS, as indicated by the lack of increase
in lipid peroxidation observed inin vitro and in vivo
experiments [21]. In previous studies [22,23], we
observed that a single intraperitoneal administration of
diamide in rats induced selective oxidation of blood
glutathione, with no evident alterations in thiol levels
in other tissues. Moreover, during diamide treatment,
Cys levels increased in plasma, indicating that Cys was
released from an extra-hematic compartment.
As invivo studies based on treatments with diamide
are essentially limited by its reaction with thiol groups
within a few seconds [24], in this paper we used slow
intravenous infusion ofthe drug. Additionally, we
monitored all physiological LMM-SH, namely Cys,
cysteinylglycine (CysGly), homocysteine (Hcys), GSH
and related disulfide forms in both blood and organs.
Disulfide forms comprise low-molecular-mass disulfides
[LMM-SS: CySS, cystinylglycine (CySSGly), homocys-
tine (HcySS) and GSSG] and mixed disulfides between
protein sulfhydryl groups and a low-molecular-mass
thiol (RSSP). This allowed us to study relatively long-
lasting effects of thiol-directed oxidative stress, and, in
particular, its influence on the inter-organ exchange of
LMM-SH.
Results
In a first set of experiments, the kinetics of plasma
thiols during and after diamide administration was
evaluated. Rats were treated for 60 min and monitored
for the next 2 h. Specifically, we measured the time
course of levels of GSH, Cys, CysGly and Hcys and
the corresponding disulfide forms calculated as the
sum of LMM-SS and RSSP (Fig. 1A–D). Values for
total disulfides are expressed as ‘thiol equivalents’, i.e.
the concentration of LMM-SH that are involved in
formation of both LMM-SS and RSSP. The level of
Cys significantly decreased by 20% during treatment
with diamide, and then returned slowly to initial
values. A parallel increase, particularly evident for
disulfides of cysteine, followed by a decrease in the
disulfide forms of both glutathione and cysteine, was
also observed, with a tendency (particularly for GSH)
to return to basal levels (Fig. 1A,B). Disulfides of
homocysteine tended to increase during the diamide
infusion and remained higher than the basal concentra-
tion during the whole experiment (Fig. 1C). Con-
versely, the CysGly redox balance was minimally
influenced by treatment with diamide (Fig. 1D). When
considering the amount of total thiols (i.e.
reduced + disulfide forms) found in plasma at each
time point during the whole course ofthe experiment
(Fig. 2), we observed that the levels of total cysteine
(and also total homocysteine, although toa smaller
extent) were markedly increased during and after
diamide infusion with respect to time zero.
The same samples were also analyzed for the LMM-
SH, LMM-SS and RSSP content in RBCs. These cells
represent the main stores of hematic GSH, which
occurs mostly (> 99%) inthe reduced form [25],
whereas Cys and other LMM-SH represent only a
minimal fraction [26]. GSH was rapidly oxidized in
RBCs mainly to mixed disulfides with proteins (GSSP)
and recovered 80% of its initial value within 2 h of
the end of treatment (Fig. 3B). Cys levels decreased
during the first part ofthe treatment (10–30 min), but
the prevailing phenomenon was a dramatic reversible
increase in both the reduced and, in particular, the
disulfide forms, mainly mixed disulfides with proteins
(CySSP) (Fig. 3A). The highest value reached for CyS-
SP was 2.5 nmol per mg Hb (i.e. 0.75 mm), and was
measured at the end of treatment with diamide. This
suggests a large, reversiblefluxofcysteinefrom other
tissues towards RBCs. Figure 3C shows the change in
total cysteine and total glutathione in RBCs with
respect to their basal levels. It is evident that the levels
of total cysteine, but not total GSH, greatly and
reversibly increased over basal levels after oxidative
stress induction by diamide. The change in total cyste-
ine concentration in both plasma and RBCs during
diamide treatment and over the subsequent 2 h is sum-
marized in Fig. 4. Values were normalized for the
hematocrit, and the level of total cysteine greatly
increased in plasma and RBCs, reaching a maximum
D. Giustarini et al. Cysteineflux under oxidative stress
FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4947
level 75 min after the start of diamide infusion. It is
evident that thecysteine that enters RBCs cannot
derive solely from plasma stores. We also monitored
the concentrations of thiols and disulfides in blood
withdrawn from rats at 75 min after the beginning of
diamide infusion (when total cysteine reached its maxi-
mum observed levels): both plasma and RBCs were
analyzed ex vivo over time. The observed concentra-
tion changes of GSH, Cys and their oxidized forms in
RBCs were similar to those observed in vivo, with thi-
ols and disulfides recovering their basal levels within
2 h after blood collection (data not shown). The
plasma Cys levels increased slightly over time, but
more evident was the increase in CySS and CySSP,
AB
CD
Fig. 1. Thiols and disulfides inrat plasma during and after diamide infusion. Time course of plasma LMM-SH (closed circles) and total disulfide
(open circles) levels during and after diamide infusion (7.73 lmolÆmin
)1
Ækg
)1
intravenously). (A) Cysteine; (B) glutathione; (C) homocysteine;
(D) cysteinylglycine. Disulfides represent the sum of low-molecular-mass disulfides and protein mixed disulfides; values are expressed as thiol
equivalents. Data are the means ± SD of four experiments. *P < 0.05 versus value at time zero; **P < 0.01 versus value at time zero.
Cysteine flux under oxidativestress D. Giustarini et al.
4948 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS
probably due to oxidation ofcysteine exported from
RBCs (Fig. 5). Little GSH was exported during this
ex vivo experiment, and the concentrations of total
GSH were measured within RBCs were constant (data
not shown).
The change in total cysteine levels was found to be
dose-dependent. In experiments performed with
various doses of diamide (dose range 1.93–9.66 lmolÆ
min
)1
Ækg
)1
), the increase inthe concentration of total
cysteine showed an almost linear dose dependence, as
shown in Fig. 6, in which the maximum total cysteine
accumulated in RBCs and plasma is plotted against
the diamide dose administered.
We further evaluated the effect of diamide infusion
on various rat organs in order to assess whether dia-
mide was also able to affect the thiol ⁄ disulfide bal-
ance of these organs, and whether cysteine efflux to
the blood was paralleled by a change inthe Cys
concentration in other tissues. Diamide, at 60 min
from the start of administration, evoked a significant
decrease inthe hepatic Cys level, followed by an
increase in both liver and kidney (after 2 h from the
end of administration), but the GSH concentration
was found to be significantly higher only inthe liver
2 h after the end of diamide administration
(Fig. 7A,B ). A slight but significant (P < 0.05)
increase inthe concentration of total disulfide forms
of GSH in heart and lung but not in other analyzed
organs was also observed (+16.2 ± 5.1% and
+20.9 ± 3.2%, respectively). Additionally, in liver,
kidney, lung and heart, a slight but significant
(P < 0.05) increase in Hcys wa seen, both at the
end of diamide infusion (i.e. 60 min) and at the end
of the experiment (i.e. 180 min) (data not shown).
These data suggest that diamide has a minimal effect
on the thiol ⁄ disulfide balance oftissues other than
blood, and did not induce evident decreases in cyste-
ine and GSH levels inthe organs analyzed.
Together, these findings prompted us to further
investigate the origin ofcysteine that enters blood.
In theory, two possible sources exist: (a) intracellular
GSH is exported and converted extracellularly to
Cys by c-glutamyl transpeptidase (c-GT) and dipep-
tidases, with cysteine being then taken up by RBCs,
or (b) methionine is first converted into Hcys, and
then, through the trans-sulfuration pathway (TSP),
to Cys. To assess these two hypotheses, we repeated
the experiments using rats pre-treated with acivicin
and ⁄ or propargylglycine. Acivicin is an inhibitor of
c-GT, thus this treatment should eliminate the frac-
tion of Cys that derives from GSH [27]. Propargyl-
glycine is an inhibitor of cystathionase, the enzyme
that forms cysteinefrom cystathionine inthe TSP; in
this case, treatment should eliminate the supply of
Cys derived from methionine [28]. Pre-treatment with
these inhibitors in control rats (administered with
saline) completely abolished the release of Cys into
blood (Fig. 8). In animals treated with diamide,
propargylglycine and acivicin significantly decreased
the fluxofcysteine towards the hematic compart-
ment by 25% and 30%, respectively. Concomitant
use of these inhibitors enhanced the inhibitory effect
on the Cys increase in blood. This suggests that
both pathways contribute tothe large cysteine efflux
from organs into the hematic compartment observed
in our experiments.
Discussion
In this study, a systemic response toa thiol-directed
oxidative insult was induced by means of slow infu-
sion of diamide inthe rat, which evoked a relatively
long-lasting oxidant stimulus. Little is known about
the effect ofoxidativestress on the distribution of
LMM-SH in extracellular fluids and their exchange
rate between tissues. The paucity of information in
this field is due, at least in part, tothe intrinsic diffi-
culty in creating reliable animal models of oxidative
stress. Acute carbon tetrachloride administration is
frequently used as a rodent model for oxidative
stress; these experiments are promising, and reports
Fig. 2. Total thiols inrat plasma during and after diamide infusion.
Time course of total plasma thiols (sum of reduced + disulfide
forms) levels during and after diamide infusion (7.73 lmolÆmin
)1
Ækg
)1
intravenously). Data are the means ± SD fromthe experiments
shown in Fig. 1. *P < 0.05 versus value at time zero; **P < 0.01
versus value at time zero.
D. Giustarini et al. Cysteineflux under oxidative stress
FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4949
have demonstrated which antioxidant parameters are
influenced by these treatments [29,30]. However, these
models are harsh, many free radicals are produced as
a result ofthe reductive bioactivation of carbon tetra-
chloride by cytochrome P450, and the animals may
suffer multi-organ damage. Thus, information on the
inter-organ trafficking of small thiols is difficult to
interpret [30,31]. In contrast, diamide is a thiol-
specific oxidizing agent that rapidly reacts with GSH
and other thiols without production of free radicals
or other ROS. Further, we performed experiments by
a slow infusion of diamide to avoid the problem that
A
C
B
Fig. 3. Thiols and disulfides inrat erythrocytes during and after diamide infusion. Time course of erythrocyte LMM-SH (closed circles),
LMM-SS (closed squares) and RSSP (closed triangles) levels during and after diamide infusion (7.73 lmolÆmin
)1
Ækg
)1
intravenously). (A) Cys-
teine; (B) glutathione. (C) Increase in total cysteine and total glutathione levels over values at time zero. Values are the sum ofthe thiol and
disulfide concentrations shown in (A) and (B), and are expressed as thiol equivalents. Data are the means ± SD of four experiments.
*P < 0.05 versus value at time zero; **P < 0.01 versus value at time zero.
Cysteine flux under oxidativestress D. Giustarini et al.
4950 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS
the diamide effect is limited to only a few seconds
once administered to rats, because of its high reacti-
vity [22,23].
The main finding of our experiments was a revers-
ible accumulation ofcysteine within theblood (Fig. 4).
Our previous studies [22,23] showed a slight increase in
hematic cysteine after a single intraperitoneal diamide
administration to rats, accompanied by oxidation of
hematic GSH; little effect was found for GSH, its
disulfide forms, and antioxidant enzyme levels in tis-
sues other than blood [23]. Conversely, a 1 h treatment
with diamide, like that performed here, evoked dra-
matic changes in hematic thiols and disulfides. More-
over, the features ofcysteine accumulation in blood
were studied in detail by separate analysis of plasma
and erythrocyte compartments. The total cysteine con-
centration inthe plasma compartment increased from
100 to 140 lm (Fig. 2) during diamide administra-
tion. In contrast, the levels of reduced cysteine did not
change (Fig. 1A). This is probably the result of two
phenomena: enhanced delivery ofcysteine into the
blood by tissues, and oxidation elicited by diamide.
The total Hcys level also showed areversible increase
(Fig. 2).
More dramatic effects were observed in erythrocytes,
in which GSH was reversibly oxidized (Fig. 3B), and,
more importantly, a large increase inthecysteine con-
centration, in both the reduced and disulfide forms,
was observed (Fig. 3A). Therefore, a high amount of
cysteine is delivered into the plasma and rapidly taken
Fig. 4. Total cysteineinratblood during and after diamide infusion.
Time course ofthe total cysteine (sum of reduced thiol + disul-
fides) increase over the levels at time zero inratblood during and
after diamide infusion (7.73 lmolÆmin
)1
Ækg
)1
intravenously). The
reported values were calculated by normalization of measured con-
centrations of total cysteinein both plasma (black bars) and erythro-
cytes (gray bars) tothe relative hematocrit value. Values are
expressed as thiol equivalents. Data are the means ± SD of four
experiments.
Fig. 5. Thiols and disulfides inrat plasma ex vivo after diamide
infusion. Ex vivo levels of plasma Cys (triangles), CySS (squares)
and CySSP (circles) after diamide infusion (7.73 lmolÆmin
)1
Ækg
)1
intravenously) and blood withdrawal. Blood was withdrawn 15 min
after the end of diamide infusion and maintained at 37 °C. Time
zero indicates the time of withdrawal. Data are the means ± SD of
three experiments. *P < 0.05 versus value at time zero;
**P < 0.01 versus value at time zero.
Fig. 6. Dose dependence ofthe maximum increase of cysteine
induced by diamide. Values indicate the increase of total Cys over
levels at time zero inratblood 75 min after the start of diamide
infusion (60 min infusion, dose range 1.93–9.66 lmolÆmin
)1
Ækg
)1
intravenously). The reported values were calculated by normaliza-
tion of measured concentrations of total cysteinein both plasma
and erythrocytes tothe relative hematocrit value. Values are
expressed as thiol equivalents. Data are the means ± SD of five
experiments.
D. Giustarini et al. Cysteineflux under oxidative stress
FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4951
up by RBCs during diamide administration (Fig. 4). In
RBCs, cysteine accumulated mainly as protein mixed
disulfides, conceivably linked tothe highly reactive
b-125 cysteine residues of hemoglobin. Analogously,
GSH is mostly oxidized to form mixed disulfides with
hemoglobin, with a minimal increase in GSSG concen-
tration [20]. This extraordinarily high accumulation of
cysteine in RBCs was found to be reversible, and Cys
reached its initial levels within 2 h after the end of
diamide administration. Cysteine is probably reduced
by the recovered GSH and exported from RBCs. In
fact, it has recently been observed that Cys-enriched
erythrocytes export cysteineina time- and concentra-
tion-dependent manner [32]. Indeed, in ex vivo experi-
ments, we observed a significant and progressive
increase in total plasma cysteine, mainly CySS and
CySSP, after diamide infusion, corresponding to the
cysteine concentration decrease within RBCs (Fig. 5).
Given that plasma is a rather oxidizing environment,
which lacks reductases [2], it is likely that the exported
Cys is oxidized both to CySS and RSSP. We cannot
exclude the possibility that a fraction of CySS may be
exported from erythrocytes. Nevertheless, our previous
data with washed RBCs indicated that, under physio-
logical conditions, human erythrocytes release cysteine
over time, mostly inthe reduced form [26]. Although
the erythrocyte transport system for Cys efflux has not
yet been identified, Yildiz et al. [32] suggested that this
process is carrier-mediated and is significantly attenu-
ated when GSH is depleted (or its synthesis is
blocked); therefore, it may require a reduced mem-
brane thiol. It is also possible that, under our experi-
mental conditions, this transport system was less
efficient during theoxidative perturbation induced by
diamide. In contrast to Cys, GSH returned to basal
levels probably as a result of thiol⁄ disulfide reactions
and NADPH-dependent reduction. De novo GSH syn-
thesis did not occur under our experimental condi-
tions, as evidenced by constant levels of total
glutathione measured invivo (Fig. 3) and ex vivo (data
not shown). This suggests that Cys loss in RBCs does
not represent a device to counterbalance de novo GSH
synthesis.
The reason for the observed Cys accumulation, first
in plasma and then in RBCs, is not clear. It is possible
that the thiol–disulfide status in plasma is finely tuned,
and the diamide-induced oxidation of Cys is compen-
sated for by efflux of GSH and Cys fromthe liver and
other organs to maintain thecysteine pool. GSH is
known to be a critical source for maintaining a steady
Cys availability as it is continuously exported out of
cells into plasma and converted to circulating cysteine
by the action of c-GT and dipeptidase (Fig. 9). Among
the various tissues, liver has been shown to play a cen-
tral role inthe GSH homeostasis, serving as the princi-
pal source ofthe GSH circulating in plasma [14,33].
The extracellular translocation of hepatic GSH and its
c-GT-dependent extracellular catabolism, resulting in
formation of plasma Cys and CySS (cysteine is rapidly
oxidized to cystine extracellularly [34]), could be a
AB
Fig. 7. Effect of diamide infusion on thiols in various rat tissues. Rats were administered diamide (7.73 lmolÆmin
)1
Ækg
)1
intravenously) for
1 h and killed 60 or 180 min after the start ofthe treatment. The levels of Cys (A) and GSH (B) in various tissues are reported. Values at
time zero are those obtained in rats treated analogously (i.e. implanted with the valve), but without any infusion. Data are the means ± SD
of four experiments. *P < 0.05 versus untreated animals; *P < 0.01 versus untreated animals.
Cysteine flux under oxidativestress D. Giustarini et al.
4952 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS
systemic response to local oxidative conditions. Never-
theless, after a 60 min diamide infusion, we observe a
significant decrease in hepatic Cys levels only (10 lm),
whereas the GSH concentration did not change
(Fig. 7). Analogously, disulfide levels did not vary to a
significant extent after diamide administration. Even
thought the whole amount ofcysteine that disappears
from the liver is released into blood, it cannot quanti-
tatively explain the dramatic rise of Cys registered
within erythrocytes. Nevertheless, we should consider
these phenomena as a dynamic process to which
numerous factors contribute in order to maintain fairly
constant thiol levels in various organs and to supply
the compounds necessary to counteract the diamide
challenge. In this context, a possible limit of our study
is that we did not record the multi-directional flux of
various thiols in ‘real time’; we could only perform our
measurements at fixed times, and these values obtained
are therefore the result of various events that are not
completely discriminated. Additionally, given the high
concentration of GSH in organs (2–10 mm), it is
evident that an increase in its release does not
necessarily produce a significant, quantifiable decrease
in its levels inthe organs themselves. Indeed, although
a 1–2% release of GSH does not induce a significant
decrease in its concentration in liver or in kidneys, it
provokes a marked effect in plasma, where its levels
(and in general the levels of all LMM-SH) are in the
micromolar range. In contrast, as disulfide levels are
very low inthe intracellular compartments ( 2000–
3000-fold lower than thiol compounds [2]), we can
assume that their contribution tothe increase in Cys is
negligible.
Additional factors may be considered to be involved
in the observed process ofcysteine accumulation
(Fig. 9). Extracellular cysteine can also derive from
intracellular stores of various tissues that can deliver it
to the plasma; the other main source is diet. During
prolonged starvation, skeletal muscle, in particular, can
deliver cysteinetothe plasma by degradation of pro-
teins [14]. Methionine from protein degradation or
from intracellular stores can also serve as a source of
cysteine through the action ofthe TSP [35]. Hcys is
synthesized from methionine by almost all cells through
the activated methyl cycle. Hcys can be reconverted
into methionine (with formation of tetrahydrofolate) or
cysteine through the TSP; alternatively, it can be
exported [36]. In our experiments, pre-treatment with
acivicin or propargylglycine, which inhibit either c-GT
or cystathionase, respectively, should block cysteine
influx totheblood during diamide infusion. In both
cases, a decrease of 25–30% inthe hematic levels of
cysteine was observed. The effect was strengthened by
concurrent administration of both inhibitors (Fig. 8).
This suggests that both sources of Cys from tissues, i.e.
GSH via c-GT and methionine via Hcys and the TSP,
are involved. Notwithstanding this, methionine avail-
ability is unlikely to be a limiting factor for plasma
cysteine fluxfrom tissues, because, under oxidative con-
ditions, many factors can contribute to deliver cysteine
to blood. Interestingly, stimulation ofthe TSP by
oxidative stress has been demonstrated previously
[35,37,38], suggesting that the redox sensitivity of the
trans-sulfuration pathway may be considered to be an
auto-corrective response that leads to an increased level
of glutathione synthesis in cells challenged by oxidative
stress. Evidence also exists indicating that c-GT may be
upregulated as adaptive response to an oxidative insult
[39]. Experiments performed using various diamide
doses (Fig. 6) showed that the process is dose-depen-
dent. Therefore, the observed phenomenon may derive
from an oxidative regulation of one or more proteins
critical tothe metabolism of cysteine, or from activa-
tion ofa systemic mechanism of Cys ⁄ GSH
delivery ⁄ uptake regulation.
Fig. 8. Effect of acivicin and propargylglycine on the cysteine
increase. Increase incysteine (sum of reduced cysteine + disul-
fides) over levels at time zero inratblood induced by diamide infu-
sion (7.73 lmolÆmin
)1
Ækg
)1
intravenously) after pre-treatment with
acivicin (open circles) or propargylglycine (closed triangles) or both
(open triangles). Data are compared with values obtained from rats
treated with diamide (7.73 lmolÆmin
)1
Ækg
)1
intravenously) without
pre-treatments (closed circles), or from rats treated with acivicin
(gray squares) or propargylglycine (closed squares) without diamide
infusion. Disulfides represent the sum of low-molecular-mass disul-
fides and protein mixed disulfides. The reported values were calcu-
lated by normalization of measured concentrations of total cysteine
in both plasma and erythrocytes tothe relative hematocrit value.
Values are expressed as thiol equivalents. Data are the
means ± SD of three experiments.*P < 0.05 versus samples trea-
ted only with diamide analyzed at the same time.
D. Giustarini et al. Cysteineflux under oxidative stress
FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4953
To summarize, our data suggest that diamide shifts
the thiol:disulfide ratio in plasma and RBCs. To coun-
teract this effect, Cys is likely to be delivered from
multiple tissues; a fraction of Cys is oxidized within
plasma by diamide, whereas another fraction enters
RBCs, where it is oxidized to CySS and mostly CySSP.
Once the diamide infusion and, consequently, the oxi-
dant stimulus, is complete, cysteine is taken up de novo
by tissues other than blood (Fig. 7A). We infer that
the x
c
)
transport system for CySS may contribute to
remove thecysteine accumulated within erythrocytes
and plasma. In fact, it has been demonstrated that this
transport system, which is widely distributed in various
organs [40,41], is upregulated under oxidative condi-
tions [42]. Therefore, it is reasonable to hypothesize
that, under our experimental conditions, CySS uptake
into various organs is enhanced, followed by CySS
reduction to Cys. Indeed, a clear increase in Cys levels
was observed 2 h after the end of diamide infusion in
the kidneys and the liver. Additionally, a significant
increase in GSH was observed inthe liver at 3 h after
the start of treatment (Fig. 7B), probably indicating
that the excess cysteine enters this organ, where it is
subsequently converted into GSH. An increase in liver
GSH after an acute increase of circulating cysteine has
been described previously [43].
Maintenance of an adequate plasma thiol–disulfide
balance appears to be fundamental. It has been dem-
onstrated that even a minimal shift inthe redox state
of either the Cys ⁄ CySS or GSH ⁄ GSSG pool (e.g. 21
and 9 mV changes, respectively, as observed in aging)
is sufficient to cause a large increase inthe oxidized
forms of intracellular proteins bearing vicinal thiols,
which can influence specific signaling pathways [44]. In
addition, it has been observed that the NMDA recep-
tor, protein disulfide isomerase, epidermal growth fac-
tor receptor and extracellular signal-regulated kinase
are modulated by the cellular and ⁄ or extracellular
redox state [45–48]. The shift inthe thiol ⁄ disulfide
redox state over the range found invivoin human
plasma is a key determinant of early events of vascular
disease development [49]. In this context, it should be
noted that Hcys levels in plasma (and in some other tis-
sues) were also reversibly increased in our experiments
by the thiol-directed oxidant, although toa minor
extent compared with Cys levels (Figs 1C and 2). This
Fig. 9. Schematic diagram showing the metabolic pathways of various physiological LMM-SH. Intracellular Cys is a key determinant in regu-
lating the kinetics of formation of c-glutamylcysteine (c-GluCys), the first step in GSH synthesis. Intracellular Cys may derive from diet, intra-
cellular stores, or methionine (Met), which is converted into Hcys by the activated methyl cycle. Hcys may then condense with serine to
produce cystathionine, which in turn may be hydrolyzed to Cys. The conversion of cystationine into Cys is catalyzed by the enzyme c-cysta-
thionase, the activity of which is inhibited by propargylglycine (PGG). Both GSH and Cys can be exported from cells into plasma, where they
undergo auto-oxidation. The liver and kidneys appear to have significant capacity for GSH efflux. Although it has been established that cells
are able to export excess GSSG, it is not yet known whether they also possess a transport system for CySS. Plasma Cys, but neither GSH
nor GSSG, can be taken up by most cells (also including RBCs). Some cell types (excluding RBCs) also possess a transport system for
uptake of CySS. Extracellular GSH can be converted into CysGly and then into Cys by the combined action ofthe membrane enzymes
c-glutamyl transpeptidase (cGT) and dipeptidases (DP). The activity of cGT is inhibited by acivicin. Alternatively, CysGly may be taken up and
converted into Cys by DPs.
Cysteine flux under oxidativestress D. Giustarini et al.
4954 FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS
effect could be indicative of diamide-induced stimula-
tion of methionine transformation into Cys, a pathway
in which Hcys represents an intermediate. Given the
well-known action of Hcys as a pro-atherogenic and
cardiovascular risk factor, the fact that diamide treat-
ment (and probably other events that perturbate the
thiol ⁄ disulfide status of plasma) may influence homo-
cysteine levels suggests that this topic deserves further
investigation.
Many authors have focused their attention on the
possible role of modulation of LMM-SH levels in dis-
ease development and ⁄ or progression. However, only a
few studies have investigated the physiological rules that
govern the distribution of LMM-SH in extracellular flu-
ids, the contribution of various tissues, the possible con-
sequence(s) of decreased ⁄ increased efflux froma single
organ because of pathophysiological conditions, and,
more importantly, the influence ofoxidativestress on
these processes. Our data clarify some of these aspects,
and suggest that thiol-directed oxidativestress consider-
ably influences the inter-organ exchange ofcysteine and
its production from methionine and ⁄ or GSH.
Experimental procedures
Animals
Sprague–Dawley male rats (400–450 g) were purchased
from Charles River Laboratories (Calco, Italy). A double
valve (model 617, 20 · 20 mm; Danuso Instruments, Milan,
Italy) was implanted in each animal; jugular and femoral
veins were cannulated for either drug administration or
blood collection, as described previously [20]. The valve
was implanted under pentobarbital anesthesia two days
before the experiment. Animals were allowed to freely move
and fed ad libitum before and during the experiments.
Rats received infusions of diamide in saline (7.73 lmol
Æmin
)1
Ækg
)1
unless otherwise specified) via the cannula
implanted inthe femoral vein. The infusion was started
immediately after blood withdrawal for time zero measure-
ments and lasted 1 h. Blood aliquots (100 lL each) were
collected through the valve connected tothe jugular vein
and immediately processed. All animal manipulations were
performed in accordance with the European Community
guidelines for the use of laboratory animals. The experi-
ments were authorized by the local ethics committee. Rats
did not show any evidence of distress or altered behavior
during the experiments.
Thiol and disulfide measurement in blood
Blood samples were collected in plastic tubes containing
K
3
EDTA. Blood was immediately centrifuged at 15 000 g
for 15 s. Aliquots of plasma (20 lL) were immediately
acidified by 1 : 1 addition ofa solution of trichloroacetic
acid (TCA, 12% w ⁄ v). After separation of proteins by cen-
trifugation at 15 000 g for 2 min at room temperature,
25 lL ofthe supernatant was brought toa pH of 8.0
using 5 lLof2m Tris, and then 0.5 lLof40mm mono-
bromobimane (mBrB; Calbiochem, Milan, Italy) dissolved
in methanol was added for LMM-SH measurement. After a
10 min incubation inthe dark, samples were acidified and
analyzed by HPLC as previously described [1]. A further
25 lL of plasma were added with 1 lLof50mm N-ethyl-
maleimide (NEM; Sigma-Aldrich, Milan, Italy,) for mea-
surement of both low-molecular-mass disulfides and protein
mixed disulfides (total disulfides). After 10 min incubation
at room temperature, NEM was extracted with four vol-
umes of dichloromethane, 2 mm (final concentration) of
dithiothreitol was added to reduce disulfides, and after
5 min samples were 1 : 1 diluted with 12% w ⁄ v TCA and
then proteins were discarded by centrifugation at 15 000 g
for 2 min at room temperature. Supernatants (30 lL) were
brought toa pH of 8.0 using 6 lLof2m Tris, and then
1.2 lL of mBrB was added. After a 10 min incubation,
samples were acidified with HCl and analyzed by HPLC as
previously reported [1]. All samples with visible hemolysis
were discarded. For determination of LMM-SS, 5 lL
aliquots of plasma (after addition of NEM, as described
above) were 1 : 3 diluted with 8% w ⁄ v TCA, and then
proteins were discarded by centrifugation at 15 000 g for
2 min at room temperature. Supernatants (10 lL) were
brought toa pH of 8.0 using 2 lLof2m Tris, then
2mm dithiothreitol (final concentration) was added. Sam-
ples were then derivatized using mBrB, and analyzed as
described above. The level of protein mixed disulfides was
calculated as the difference between total disulfides and
LMM-SS.
The RBC pellet was washed three times with ice cold
Na
+
⁄ K
+
phosphate-buffered saline (pH 7.4) containing
5mm glucose for LMM-SH analyses or 5 mm glucose
and 80 mm NEM for disulfide analyses, and hemolyzed
using 10 volumes of 10 mm Na
+
⁄ K
+
phosphate buffer, pH
7.4. Aliquots ofthe hemolysate were used for LMM-SH,
total disulfide or LMM-SS determinations as described
above.
For ex vivo experiments, after 75 min fromthe start of
diamide infusion, 600 lL ofblood was withdrawn from
the valve (K
3
EDTA was used to prevent blood clotting)
and maintained at 37 °C under gentle gyratory shaking for
analyses of thiols and related disulfides. At the indicated
times, aliquots ofblood were centrifuged at 15 000 g for
15 s. Thiols and disulfides were analyzed in both plasma
and RBCs as described above.
All HPLC analyses were performed by using a Spheri-
sorb C18 column (4.6 mm internal diameter · 250 mm
column length) (Varian Inc, Palo Alto, CA) by means of an
Agilent series 1100 HPLC (Agilent Technologies, Milan,
Italy) equipped with a fluorometric detector.
D. Giustarini et al. Cysteineflux under oxidative stress
FEBS Journal 276 (2009) 4946–4958 ª 2009 The Authors Journal compilation ª 2009 FEBS 4955
[...]... whereas acivicin was administered in two doses (each 10 mgÆkg)1 intraperitoneally) 2.5 and 1 h before starting diamide infusion In experiments with control rats, only saline was infused For the assessment of thiols and disulfides inrat tissues, animals were anesthetized with sodium pentobarbital (60 mgÆkg)1), blood was withdrawn from abdominal aorta, and then tissues were rapidly collected All organs.. .Cysteine flux under oxidativestress D Giustarini et al Thiol and disulfide measurements inrattissues Treatments with acivicin (a c-GT inhibitor) and propargylglycine (a cystathionine c-lyase inhibitor) were performed as described by Zalups and Lash [50] and Shinozuka et al [51], respectively Propargylglycine (100 mgÆkg)1 intraperitoneally) was administered 5 h before starting diamide infusion,... (2001) Novel approach for the determination ofthe redox status of homocysteine and other aminothiols in plasma from healthy subjects and patients with ischemic stroke Clin Chem 47, 1031–1039 14 Ookhtens M & Kaplowitz N (1998) Role ofthe liver in interorgan homeostasis of glutathione and cyst(e)ine Semin Liver Dis 18, 313–329 15 Bannai S (1984) Transport of cystine and cysteinein mammalian cells Biochim... described above Statistics Data are expressed as means ± SD Differences between means were evaluated using one-way analysis of variance (ANOVA) A value of P < 0.05 was considered statistically significant Acknowledgements This work was supported by grants fromthe Fondazione Monte dei Paschi di Siena, by FIRST (Fondo Interno Ricerca Scientifica e Tecnologica, University of Milan) 2007, and by the Fondazione Ariel,... Milzani A & Giustarini D (2006) Oxidized forms of glutathione in peripheral blood as biomarkers ofoxidativestress Clin Chem 52, 1406–1414 22 Di Semplicio P, Giannerini F, Giustarini D, Lusini L & Rossi R (1998) The role ofcysteineinthe regulation ofblood glutathione–protein mixed disulfides in rats treated with diamide Toxicol Appl Pharmacol 148, 56–64 23 Giannerini F, Giustarini D, Lusini L, Rossi... & Jacobsen DW (2001) Relative roles of albumin and ceruloplasmin inthe formation of homocystine, homocysteine cysteine- mixed disulfide, and cystine in circulation J Biol Chem 276, 46896– 46904 Mosharov E, Cranford MR & Banerjee R (2000) The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes... Oxidativestress as a signal to up-regulate gamma-cystathionase inthe fetal -to- neonatal transition in rats Cell Mol Biol 53, 1010–1017 Liu RM, Shi MM, Giulivi C & Forman HJ (1998) Quinones increase c-glutamyl transpeptidase expression by multiple mechanisms inrat lung epithelial cells Am J Physiol 274, L330–L336 Christensen HN (1990) Role of amino acid transport and countertransport in nutrition and... Carlson BA, Cheng PY, Park Y, Cotsonis GA, Accardi CJ & Jones DP (2007) Diurnal variation in glutathione and cysteine redox states in human plasma Am J Clin Nutr 86, 1016–1023 18 Dalle-Donne I, Rossi R, Colombo R, Giustarini D & Milzani A (2006) Biomarkers ofoxidative damage in human disease Clin Chem 52, 601–623 19 Dalle Donne I, Giustarini D, Colombo R, Milzani A & Rossi R (2005) S-glutathionylation... Stipanuk MH (2003) Cysteine is the metabolic signal responsible for dietary regulation of hepatic cysteine dioxygenase and glutamate cysteine ligase in intact rats J Nutr 133, 2697–2702 29 Griffiths HR, Møller L, Bartosz G, Bas A, BertoniFreddari C, Collins A, Cooke M, Coolen S, Haenen G, Hoberg AM et al (2002) Biomarkers Mol Aspects Med 23, 101–208 30 Kadiiska MB, Gladen BC, Baird DD, Germolec D, Graham... Carcinog Ecotoxicol Rev 25, 185–209 Yildiz D, Uslu C, Cakir Y & Oztas H (2006) l -Cysteine in ux and efflux: a possible role for red blood cells in regulation of redox status ofthe plasma Free Radic Res 40, 507–512 Ookhtens M, Mittur AV & Erhart NA (1994) Changes in plasma glutathione concentrations, turnover, and disposal in developing rats Am J Physiol 266, R979– R988 Sengupta S, Wehbe C, Majors AK, . Oxidative stress induces a reversible flux of cysteine from
tissues to blood in vivo in the rat
Daniela Giustarini
1
, Isabella Dalle-Donne
2
, Aldo. and whether cysteine efflux to
the blood was paralleled by a change in the Cys
concentration in other tissues. Diamide, at 60 min
from the start of administration,