From the Department of Neuroscience, Pharmacology
Unit, Via A. Moro 4, University of Siena, 53100 Siena, Italy and the
§ Laboratory of Biochemistry and Biophysics of the
Cytoskeleton, Department of Biology, Via Celoria 26, University
of Milan, 20133 Milan, Italy
Received for publication, June 14, 2000, and in revised form, September 20, 2000
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ABSTRACT |
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The effect of oxidants, electrophiles, and NO
donors in rat or human erythrocytes was analyzed to investigate the
influence of protein sulfhydryl groups on the metabolism of these thiol reactants. Oxidant-evoked alterations in thiolic homeostasis were significantly different in the two models; large amounts of glutathione protein mixed disulfides were produced in rat but not in human erythrocytes by treatment with hydroperoxides or diamide. The disappearance of all forms of glutathione (reduced, disulfide, protein
mixed disulfide) was induced by menadione only in human erythrocytes.
The treatment of rat red blood cells with electrophiles produced
glutathione S-conjugates to a much lower extent than in
human red blood cells; GSH was only minimally depleted in rat red blood
cells. The NO donor S-nitrosocysteine induced a rapid transnitrosation reaction with hemoglobin in rat erythrocytes producing
high levels of S-nitrosohemoglobin; this reaction in human
red blood cells was negligible. All drugs were cleared more rapidly in
rat than in human erythrocytes. Unlike human Hb, rat hemoglobin
contains three families of protein SH groups; one of these located at
position Glutathione is the major low molecular weight thiol in mammalian
cells where it constitutes the most important antioxidant defense. Its
action is usually favored by ubiquitous enzymes
(e.g. glutathione S-transferases, glutathione
peroxidase). GSH also regenerates other important defensive resources
(e.g. vitamins E and C) and directly participates in the
destruction of reactive oxygen species (1). The alkylation of
glutathione by electrophilic reagents and the reduction of chemically
reactive oxidant species are biological functions associated with the
protection of SH groups of critical cellular macromolecules (2).
The cell concentration of protein SH groups, ranging from 10 to 30 mM, is larger by far than that of GSH (2-10
mM) (3); however, the metabolic role against electrophiles
and oxidants is thought to be rather marginal. In fact, the
modification of PSH1 has been
usually considered only a potential damaging reaction, even if some
authors suggested that chemical alteration of some cysteine residues
may have a protective or regulatory role (4, 5).
The participation of PSH in quantitatively important reactions is
proportional to their concentration and reactivity. The intrinsic
reactivity of PSH is dependent on the pKa value (the
thiolate anion is much more reactive than the undissociated form) and
its accessibility (structural and conformational features). In fact,
some proteins such as albumin (6) with relatively low
pKa values have an apparent reactivity much lower than that of GSH. It follows from this that PSH may have a wide range
of apparent reactivity that spans 4 orders of magnitude (7). This fact
has abrogated any efforts to understand the effective defensive
contribution of PSH. More recently, the attention on PSH as important
modulators of the intracellular redox state has been renewed and
emphasized (5, 8, 9), and reports of PSH having reactivity very similar
to or greater than that of GSH are increasing.
Recently, the problem has become more interesting because a large
number of papers have focused on the importance of the cooperation between GSH and protein SH group, in particular hemoglobin, in nitric
oxide transport, and targeting (10, 11). Thus, the role of PSH and GSH
as similar cooperating groups in the metabolism of electrophiles,
oxidizing agents, and nitric oxide remains an open question.
Our previous studies have demonstrated that rat hemoglobin possesses
reacting cysteines located on the To verify whether and by which mechanism, the reactivity of rat Hb
cysteines leads to an unusual metabolism of some thiol reactants, we
analyzed the effect of three different drug categories, oxidants,
electrophiles, and NO donors on human and rat erythrocytes. Among the
oxidants we used were drugs that react with GSH either enzymatically
(via GSH-peroxidase, i.e. hydroperoxydes) or
nonenzymatically (diamide, menadione); similarly, electrophiles that
conjugate with GSH by glutathione S-transferases catalysis
or spontaneously (13) were tested. The membrane-permeable NO donor
S-nitrosocysteine was also used to evaluate the extent of
transnitrosation reactions carried out by PSH.
Chemicals--
Glutathione, glutathione disulfide, and
glutathione reductase were obtained from Roche Molecular Biochemicals;
HPLC grade chemicals were from BDH; BCNU was from Bristol-Myers
Squibb (Dublin, Ireland). Diamide (diazenedicarboxylic acid
bis(N,N-dimethylamide)), menadione
(2-methyl-1,4-naphthoquinone), tert-butyl hydroperoxide, human serum albumin, and all other chemicals of analytical grade were
from Sigma.
Blood Collection--
Male Wistar rats (2 months old, 300-g body
weight) were purchased from Charles River (Como, Italy). Blood was
collected from the abdominal aorta under anesthesia with diethylether.
Human blood was obtained from healthy volunteers (30-45 years old). For all blood samples K3EDTA was used as anticoagulant.
Red Blood Cell Treatment--
For in vitro treatments
each blood sample was washed with PBS containing 10 mM
glucose and was adjusted to a hematocrit value of 37.5% with the same
buffer. Red blood cells samples were placed in plastic tubes, incubated
at 37 °C in a thermostatic rotating (100 rev/min) bath, and then
exposed to the different substances.
For the in vivo treatment BCNU was infused via PE-50 tubing
cannulated into the femoral vein, whereas blood was collected from a
PE-50 tubing cannulated into the jugular vein. Both tubes were
connected to a double valve (model 617, 20 × 20 mm, Danuso Instruments, Milano, Italy). Valves and tubing were implanted 2 days
before the experiment, under pentobarbital anesthesia (50 mg/kg body weight).
GSH, GSSG, and GSSP Determinations in Blood Samples--
Blood
aliquots were deproteinized by the addition of four volumes of 5%
trichloroacetic acid; GSH and GSSG were then determined on the clear
supernatant. GSH was assayed enzymatically, using CDNB and glutathione
S-transferases (12); GSSG was assayed enzymatically at 340 nm by the procedure of Klotzsch and Bergmeyer (14).
To determine GSSP concentrations, acid precipitated proteins were
washed thoroughly with the precipitating solution until no trace of
soluble GSH or GSSG was detected. The pellets were then resuspended and
brought to an alkaline pH (pH 7.5-8.0 for samples of rat red blood
cells and pH 12.0 for the human erythrocytes). The alkaline pH (12.0)
is used in human samples because only under these conditions all mixed
disulfides are released; for rat samples slightly alkaline buffers were
used (pH 7.5-8.0), but similar results are obtained at pH 12.0. Under
these conditions GSH is released via an SH/SS exchange reaction as
previously described by Rossi et al. (15). The amount of
released GSH was assayed enzymatically in the supernatant (see above).
Hemoglobin Preparation--
Rat hemoglobin was purified by
crystallization from stroma-free hemolysate by the method of
Condò et al. (16). Human hemoglobin was prepared using
the method of Riggs (17). Hemoglobin stocks were prepared and
maintained under N2. The amount of oxidized hemoglobin was
checked spectrophotometrically before each experiment on the basis of
the 576/541 nm absorbance ratio (18).
Hemoglobin Sulfhydryls Titration and Determination of the
Reactivity Constants (k2)--
PSH of hemoglobin were
assayed spectrophotometrically with DTNB at 450 nm ( Glutathione Reductase Activity--
Glutathione reductase
activity determination was carried out at room temperature on
hemolysate, previously passed through a Sephadex G-25 column, according
to standard methods (19).
Nitrosothiols--
Cys-NO was freshly prepared by combining
equimolar concentrations of 200 mM Cys in 0.75 N HCl and 200 mM potassium nitrite in the
presence of 0.1 mM diethylenetriaminepentaacetic acid. After 3-5 min, the mixture was neutralized with 1 M Tris.
Cys-NO and nitrosoproteins titration was carried out after
decomposition of the S-NO bond with Hg2+ and is based on
the colorimetric determination of
NO Glutathione Conjugate and Free Drug
Determinations--
Glutathione conjugates and free substances were
determined on deproteinized samples (final concentration, 5%
trichloroacetic acid) by HPLC or by spectrophotometry. Glutathione-CDNB
and glutathione-EA conjugates were measured by evaluation of the peak
at 340 and 290 nm absorbance, respectively; free CDNB and EA were
titrated by end point reactions after the addition of glutathione
transferases and an excess of GSH. All other drugs and their adducts
with GSH were measured by HPLC. Briefly, deproteinized samples were
loaded onto a Sephasil C18 (250 × 4.6 mm) column (Amersham
Pharmacia Biotech) and eluted by the application of a linear gradient
of methanol: 0-10 min 20% methanol 80% acetate buffer (100 mM, pH 4.5), 10-30 min linear gradient 20-100% methanol.
For HPLC determination of glutathione conjugates of BCNU, a BioRad
Biosil NH2 column (250 × 4.6 mm) was used;
deproteinized samples were treated with iodoacetic acid for 45 min ( final concentration, 15 mM) at neutral pH and
derivatized with an alcoholic 1.5% solution of
2,4-dinitrofluorobenzene (1:1 for 3 h). Samples were eluted by the
application of a linear gradient of 0.5 M acetate buffer, pH 4.7, after a 10-min isocratic phase (0-10 min with 20% acetate, 80% methanol; 10-30 min with 20-90% acetate). An Hewlett Packard HPLC Series 1100 equipped with diode array detector was used. All
spectrophotometric determinations were carried out with a JASCO UV550 apparatus.
Oxidizing Agents--
Treatment of human and rat erythrocytes with
oxidizing substances evidenced remarkable differences between the two
species; hydrogen peroxide or organic hydroperoxides
(tert-butyl hydroperoxide) produced a rapid oxidation of GSH
to GSSG (Fig. 1), both in human and rat
red blood cells. However, rat erythrocytes were also characterized by a
subsequent, rapid formation of glutathione-protein mixed disulfides
(GSSP). The initial thiolic homeostasis was completely restored within
1 h of drug exposure. Diamide (a specific oxidant for thiol
groups) rapidly increased GSSG levels in human red blood cells;
conversely, it produced GSSP but not GSSG in rat erythrocytes.
Fig. 1 shows the results of the treatment of rat and human red blood
cells with menadione (vitamin K3). Menadione is a quinone reagent that produces oxidative modifications in biological systems, mainly through redox cycling reactions with oxygen (menadione binds to
thiol groups to form a conjugate able to redox cycle like menadione
itself) (21). Human red blood cells were essentially characterized by a
rapid GSH disappearance with a negligible (if any) production of GSSG
and GSSP. In contrast, in rat erytrocytes, menadione immediately
produced large amounts of GSSG, which in turn were transformed into
GSSP. In both cases, human and rat red blood cells were not able to
restore initial levels of GSH. Total glutathione (GSH + GSSG + GSSP)
was greatly decreased in human samples, whereas only a slight decrease
was found in rat erythrocytes.
Electrophilic Agents--
Glutathione alky(ary)lation by
electrophilic reagents is considered an important metabolic pathway of
many drugs and their intermediates. The formation of these conjugates
is a detoxification reaction because the glutathione-electrophile
adduct is usually less toxic than the parent molecule; after its
formation, the conjugate is exported to the extracellular milieu by
specific transporters (22). GSH alky(ary)lation is usually catalyzed by
GSH S-transferases, a ubiquitous class of isoenzymes, which can be divided into four families (
Rat and human red blood cells were treated with two known substrates of
GSH S-transferases (CDNB and EA) and with other reagents that conjugate GSH nonenzymatically (N-ethylmaleimide) or
after decomposition into subproducts (BCNU). After drug addition (1 mM, approximately at a molar ratio of 1:1 with GSH), a
rapid increase in intraerythrocytic GSH conjugates (Fig.
2A) was detected in all
samples, followed by their export to the extracellular medium (Fig.
2B). However, remarkable differences were evident between rat and human red blood cells. In particular, in the rat, the peak of
intraerythrocytic GSH conjugates was always much lower than that
obtained in human samples; similarly, the amount of exported conjugate
was considerably lower than in human erythrocytes.
In parallel, GSH was almost totally depleted in human erythrocytes
treated with EA, CDNB, BCNU, or N-ethylmaleimide, but not in
rat red blood cells; a percentage varying from 65 to 95% of initial
GSH was still present in rat erythrocytes after 4 h of exposure
(Table I).
NO Donors--
A large interest in the biochemistry of SH groups
has been stimulated by the discovery that they can have an active role
in nitric oxide metabolism and targeting through the formation of S-nitrosothiols. These molecules have been shown to undergo
exchange reactions with low and high molecular weight thiols, reactions of primary importance in the final effect of nitric oxide itself (24).
In Fig. 3, the influence of a treatment
of red blood cells with S-nitrosocysteine on the levels of
intracellular protein S-nitrosothiols is shown.
S-Nitrosocysteine is a compound able to cross plasma membranes rapidly and to exchange NO+ group with other
thiols. The kinetics of these reactions are governed by the reactivity
and concentration of various thiol groups. Only slight variations in
S-nitrosoprotein levels were found when human erythrocytes
were used; conversely, under the same conditions rat red blood cells
rapidly produced high levels of S-nitrosohemoglobin.
Rates of Drug Removal--
Among the substances used in the
present work, some are known to be exclusively metabolized (at least in
red blood cells) through conjugation with SH groups, whereas
hydroperoxides (25) and menadione (26) can also react with
Fe2+ of the Hb heme group. In Table
II the t1/2 and the
time required for complete disappearance of free drugs (we did not
consider those metabolized also by heme groups) are reported. Rat red
blood cells removed all substances at a significant higher rate; within
a few minutes (1-5 min) all substances were in fact under the
detection limit, whereas high levels of drugs were maintained in human
erythrocytes for a long time. BCNU (the rate-limiting step is given by
its spontaneous decomposition) was metabolized more slowly by both
systems; however, its t1/2, as previously reported
(27), was half in rat erythrocytes compared with that in human red
blood cells.
Hemoglobin SH Groups--
The reactivity of various SH groups was
tested with the common sulfhydryl titrant 5,5' dithio-bis-nitrobenzoic
acid. Rat hemoglobin (Fig. 4) was
characterized by a rapid reaction when compared with other low
molecular weight thiols or protein cysteines (human Hb, human serum
albumin, GSH, N-acetylcysteine); the shape of the titration
curve is essentially biphasic, showing an initial burst phase followed
by a slower increase in absorbance. The most important feature is that
some rat hemoglobin sulfhydryls, located at the
Rat and human red blood cells used in previous experiments (Figs. 2 and
3) were also assayed for Hb-SH content; in Table
III data obtained at the end of the
experiment (4 h) are reported. Rat Hb-SH Glutathione is commonly considered to be the first line of defense
against oxidizing molecules; it also plays an important role in the
detoxification pathways of electrophilic drugs (29). Many models show
how GSH depletors may initiate irreversible damage only after a
significant decrease (greater than 70-80%) of tissue GSH. The
involvement of the SH groups of proteins in GSH-typical reactions has
been regarded as marginal, in quantitative terms, and as deleterious to
protein structure and function. Even if protein thiols are usually more
concentrated than GSH in cellular pools, their reactivity is usually
2-4 orders of magnitude lower; this allows GSH to exhibit its
protective role (30). The catalysis of a wide number of conjugation
reactions by glutathione transferases provides a further protection of
protein SH groups by GSH (2).
The reaction rate of small thiols (i.e.
N-acetylcysteine, cysteine, or glutathione) with titrating
molecules is usually well correlated with the pKa of
the sulfydryl group (which normally lies between pH 8.5 and 9.5).
However, for PSH, accessibility as well as the dissociation state of
the sulfur must be considered (3). Various protein SH groups have been
identified as "fast reacting," for example, SH of cysteine
proteases (papain) (31), or SH involved in the catalysis of glycolytic
reactions (glyceraldehyde-3-phosphate dehydrogenase) (32); however,
their overall quantitative contribution to the metabolism of
electrophiles or oxidants is clearly marginal.
Blood is characterized by the presence of high concentrations of Hb
(8-10 mM, as monomer), a value significantly larger than GSH (0.8-1 mM). It is well known that human Hb has only
one titrable pair of thiols per tetramer (Cys(F9)93 Rat hemoglobin is known to possess extra reactive SH groups compared
with human Hb; cysteines located at Comparison (Fig. 4) of the reaction kinetics of various SH groups with
DTNB demonstrated how rat Hb is essentially characterized by two
different families of thiols. One family has a reaction rate 1 and 3 orders of magnitude greater than small thiols and other protein thiols
(e.g. human Hb Cys- This has proved the existence of thiols on rat Hb possessing an
unusually high reactivity. The task is now to verify whether this
anomaly can in any way influence the overall metabolism of some agents.
Usually, treatment with oxidizing agents (e.g. organic
hydroperoxides) leads to the oxidation of GSH into GSSG. After drug reduction, the system recovers the initial levels of GSH. If the oxidative stress is severe, a SH-SS exchange reaction between GSSG and
PSH can take place (34, 35) to form protein glutathione mixed
disulfides. This reaction is more effective in the presence of highly
reactive SH groups (34).
From the data shown in Fig. 1, it can be readily deduced how rat red
blood cells, by means of fast reactive Hb thiols, are able to form
large amounts of GSSP; in contrast, only GSSG is generated in human
erythrocytes. The differences between the two species are more evident
when diamide or menadione are used. In the first case, GSSP, but not
GSSG, were produced by rat red blood cells. This was due to the rapid
reaction of diamide with Cys- Menadione-evoked alterations of GSH are rather complex. Because this
drug is able both to oxidize and conjugate, the interpretation of its
overall action is not easy. The negligible production of GSSG and GSSP
in human red blood cells and, in contrast, the generation of both GSSG
and GSSP in rat red blood cells, after menadione treatment, can be
interpreted in the light of the presence of fast reacting SH in the
rat. Menadione is likely transformed into semiquinone by reaction with
Fe2+ of Hb (26); the semiquinone is probably an
electrophile stronger than menadione itself and rapidly conjugates with
GSH. This may explain the disappearance of all forms of GSH in human
red blood cells (reduced, disulfide, mixed disulfide). On the other
hand, rat hemoglobin can intercept the semiquinone by its Cys- The conjugation activity of rat Hb toward electrophiles is also
demonstrated in Fig. 2; in all time courses of human erythrocytes, a
rapid formation of glutathione conjugates is evident, followed by its
export to the extracellular milieu. Rat erythrocytes metabolized the
drugs by GSH conjugation to a remarkably lower extent; in fact, both
intracellular and extracellular levels of conjugates were far lower
than that of the corresponding human samples.
In addition, GSH levels were close to 0 in all human red blood cells
samples after treatments, whereas in rat erythrocytes most of the GSH
was preserved (Table I). Rat erythrocytes usually quickly metabolized
all compounds (Table II) unlike human red blood cells (high levels of
free drug persisted for minutes to hours). Our data suggest that rat Hb
can play a role in the metabolism of such substances by its fast
reacting Cys- An example confirming this hypothesis can be obtained by data on BCNU
(Fig. 6). BCNU is a cytostatic drug that
belongs to the group of DNA alkylating and cross-linking agents (39).
Additional mechanisms of BCNU action have been proposed (27), notably
the inhibition of glutathione reductase. This further mechanism of action is considered to contribute both to the desired effects and the
toxicity of the drug (27). Erythrocyte GR activity has been shown to be
reduced to 10% of the initial value in patients 10-30 min after BCNU
administration (40).
125 is directly implicated in the metabolism of thiol
reactants. This is thought to influence significantly the biochemical,
pharmacological, and toxicological effects of some drugs.
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DISCUSSION
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chain at position 125 (12). These
cysteinyl residues, given their reactivity and concentration in rat
blood, competing and cooperating with GSH, may influence the
pharmacokinetics, toxicity, and action of many drugs or their metabolites.
EXPERIMENTAL PROCEDURES
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mM = 7.0 mM
1 cm
1): hemoglobin was
adjusted to pH 7.4 with 0.2 M sodium/potassium phosphate
buffer, and then the reaction was started by addition of DTNB (final
concentration, 0.2 mM). Fast reacting SH groups were
titrated by means of an Applied Photophysics MV 17 stopped flow
apparatus; samples of hemoglobin in 0.2 M phosphate buffer, pH 7.4, were rapidly mixed with equal volumes of a solution containing 0.4 mM DTNB in 0.2 M phosphate buffer, pH 7.4. Data were fitted to a single exponential curve for the human protein
and a three exponential curve for those of rat using the SigmaPlot,
version 2.01 (Jandel Scientific).
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Fig. 1.
Treatment of human and rat red blood cells
with oxidants. Time course of GSH, GSSG, and GSSP in human and rat
red blood cells (37.5% Hct in PBS) after treatment with various
oxidants (final concentration, 2 mM). At specified times
aliquots were withdrawn, and after acid precipitation GSH and GSSG were
measured in the clear supernatant; pellets were analyzed for the GSSP
content. S.D. values were omitted for clarity. The number of replicate
experiments was four. When hydrogen peroxide was used cells were
pretreated with 1 mM NaN3 to inhibit
catalase.
, µ,
, and
encoded by
different genes) (23).
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Fig. 2.
Treatment of human and rat red blood cells
with electrophilic agents. Time course of GSH conjugates after
treatment of rat (closed symbols) or human (open
symbols) red blood cells (37.5% Hct in PBS) with 1 mM
(final concentrations) of CDNB ( ,
), EA (
,
),
N-ethylmaleimide (
,
), and BCNU (
,
) at
37 °C. A, intraerythrocytic levels of conjugates.
B, extracellular levels of conjugates. At specified times,
red blood cells were pelleted by centrifugation, and proteins were
removed by acidification and centrifugation. In the supernatant,
conjugates were measured by spectrophotometry or HPLC as specified in
methods. S.D. values were omitted for clarity. The number of replicate
experiments was 3. BCNU does not react directly with thiols, thereby in
the plot we refer to the 2-chloroethylisocianate (produced by BCNU
decomposition) conjugate with GSH.
GSH concentration in rat or human red blood cells (37.5% Hct in PBS)
after 4 h of treatment with various drugs (1 mM,
37 °C)
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Fig. 3.
Treatment of human and rat red blood cells
with Cys-NO. Rat ( ) and human (
) red blood cells (37.5% Hct
in PBS) were treated with Cys-NO (final concentration, 1 mM) at 37 °C. At specified times, cells were pelleted by
centrifugation and hemolyzed by the addition of 10 volumes of water;
the samples were then passed through G25 columns, and nitrosothiols
were measured on the protein fraction. The number of replicate
experiments was three.
Half-life and time required for complete removal of various drugs
(diamide, 2 mM; all other drugs, 1 mM;
37 °C) in rat or human red blood cells (37.5% Hct in PBS)
125 position (12),
are far more reactive than GSH itself; other cysteines, in positions
13 and
93, are far less reactive. Titration of these cysteines
corresponds to the slower phase of the curve. Human Hb possesses only
one slow reacting cysteine at position
93 (28). The second order
rate constant (k2) of rat Hb Cys-
125 is about
1 order of magnitude greater than that of GSH and other low molecular
weight thiols and 3-4 orders of magnitude greater than the
k2 of other protein cysteinyl residues.
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Fig. 4.
Titration of various SH with DTNB.
Thiols (hemoglobins, 10 µM; albumin, 20 µM;
GSH and N-acetylcysteine, 10 µM) in 0.2 M phosphate buffer, pH 7.4, were titrated with DTNB (final
concentration, 0.2 mM) at 25 °C. A, rat
hemoglobin; B, GSH; C,
N-acetylcysteine; D, human serum albumin;
E, human hemoglobin.
93 and
13, as well as
human Hb-SH
93, were modified negligibly by the treatments; in
contrast, rat Hb-SH
125 (Table III and Fig.
5) were significantly decreased (the
depletion of Cys-
125 is highlighted by the lower absorbance reached
by tracings within 1 s). Depletion of rat Hb Cys-
125 elicited
by drug addition, ranged from 650 to 850 µM and was
consistent with the amount of drug, which was not found as glutathione
conjugate. A dose-dependent depletion of Hb Cys-
125 was
also induced by the oxidants, diamide, tert-butyl
hydroperoxide, hydrogen peroxide, and menadione (data not shown).
Levels of Hb SH groups in rat or human red blood cells (37.5% Hct in
PBS) after 4 h of treatment with various drugs (1 mM,
37 °C)
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Fig. 5.
Titration of Rat Hb-SH groups after treatment
of red blood cells with electrophilic agents. Rat red blood cells
(37.5% Hct in PBS) were treated for 4 h with 1 mM
CDNB, EA, or BCNU at 37 °C. Samples were then hemolyzed and passed
through G25 columns, and protein content was adjusted to the same final
concentration (with phosphate buffer) and titrated with DTNB.
A, control; B, BCNU; C, CDNB;
D, EA.
DISCUSSION
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) and that its
reactivity is also dependent on the quaternary conformation, being
slower in the deoxy state (10). Cys-
93 of human Hb is shielded by a
hydrogen bond between Asp-94 and Glu-90 (28) and is also influenced by
the vicinity of the terminal carboxyl group.
125 and
13 (33) positions
were identified as additional potentially reactive cysteinyl groups.
Cysteine
125 was shown to have a low pKa, a high
accessibility, and consequently a strong reactivity (12). Moreover, its
reactivity is not influenced by allosteric changes in the protein. This
low pK is due to a hydrogen bridge between SH of Cys-
125 and
Ser-123, where SH is the hydrogen donor.
93), respectively.
125 of rat Hb to form an intermediate,
which is then cleaved by a molecule of GSH to form mixed disulfide with
hemoglobin (36). The irreversible GSSP production in rat red blood
cells after diamide treatment is likely to be due to the trapping of
all available GSH into GSSP; in fact, the reduction of protein mixed
disulfides is catalyzed by thioltransferase, which needs GSH as a
cofactor (37).
125. The thiolic conjugates of menadione are known to redox cycle (38) (like
menadione itself), and this is evident by subsequent oxidation of GSH
in rat red blood cells. We can also speculate that the conjugate of
menadione with Cys-
125 can redox cycle more efficiently than the GSH
menadione adduct.
125 (Table III and Fig. 5) with a possible influence on
the biochemical, pharmacological, and/or toxicological activity of many drugs.
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Fig. 6.
Activity of erythrocytic glutathione
reductase after treatment with BCNU. Human (open
symbols) and rat (closed symbols) red blood cells
(37.5% Hct) were treated with different concentrations of BCNU
(A). After 120 min, samples were hemolyzed by the addition
of 10 volumes of water and assayed for GR activity. In vivo
infusion (B) of BCNU (200 mg/m2, 45-min infusion
time) through the valve connected with femoral vein. At specified times
blood aliquots were withdrawn from the valve connected to the jugular
vein, hemolyzed and assayed for GR activity. The number of replicate
experiments was three.
After incubation with BCNU (dose range, 2-200 µM), the
residual activity of GR showed little variations in rat red blood cells even when high concentrations of BCNU were used. In contrast, a
dramatic decrease was observed in human erythrocytes. The inhibition is
due to the binding of the BCNU decomposition product,
2-chloroethylisocianate, to the SH group of the catalytic site of
glutathione reductase (27). Rat Hb can intercept this molecule (via
Cys-125), thus protecting GR from inhibition (IC50
values were 350 ± 25 and 5.5 ± 2.0 µM for rat
and human red blood cells, respectively). Furthermore, the in
vivo treatment of the rat with BCNU leads to minimal, if any,
changes in GR activity. Even if higher doses than that generally used
for chemotherapy (which was shown to inhibit GR up to 90% hematic GR)
(40) were administered to rats (Fig. 6B), no variations in
rat hematic GR were found.
Nitric oxide is a signaling molecule that has captured much the
attention of researchers. NO has a short half-life in vivo and the existence of more stable transport forms of NO has been postulated (41). NO and NO derived species (NOx) can react
with protein and nonprotein thiols forming nitrosothiols.
S-nitroso derivatives of glutathione, cysteine, hemoglobin,
bovine serum albumin, and other protein or nonprotein thiols are
potent, fast acting vasodilators as well as strong inhibitors of
platelet aggregation (24). Stamler and co-workers (10, 11) reported
that Hb can act either as a sink or as a donor of nitric oxide
depending upon its quaternary state, with a pivotal role in the
regulation of blood vessel tone. In these reports they suggest that
exchange reactions between low molecular weight nitrosothiols and SH
groups of Hb play an important role in mediating nitric oxide effects in the bloodstream. In describing their model, the authors pointed out
that Cys-93 of human Hb is involved in red blood cells nitric oxide
handling and that a rapid exchange reaction between
S-nitrosocysteine and Hb-SH groups takes place. In some
experiments however, rat instead of human red blood cells were used.
Our data demonstrated that fundamental differences exist among the two
systems, with only rat Hb being rapidly nitrosylated. After treatment
with S-nitrosocysteine (Fig. 3) the intracellular levels of
S-nitroso hemoglobin rose rapidly in rat red blood cells
(0.22 NO mol/mol tetramer of Hb, after 2 min), whereas minimal
variation was found in human erythrocytes (0.007 NO mol/mol tetramer of
Hb, after 5 min); the use of rat instead of human blood could lead
thereby to some misinterpretation of results representing a poor and
irrilevant model as to human studies.
In conclusion, data obtained from our work suggest that when the rat is
used as an animal model, some attention must be paid to the unusual
behavior of its Hb-SH groups. Because of the reactivity of these
cysteinyl residues, the rat is an interesting but complicated model in
which to study the pharmacological and toxicological action of some
drugs. The fact that rat Hb possesses six reactive cysteines/tetramer,
makes rat blood per se a bad model for some biochemical/physiological interpretations (i.e.
nitrosothiols action). Furthermore, if we consider that cys 125 is
extremely reactive (about 3 orders of magnitude greater than human
93), this makes rat red blood cells able to metabolize some
thiol-reacting substances mainly through conjugation/oxidation of its
cysteine
125.
Wistar strain male rats were used for all the experiments reported in this work; however, also other strains of rat (e.g. Harlan, Sprague-Dawley) behave similarly (not shown). Also rats of different ages and body weights were studied (age, 6-9 months; body weight, 450-550 g), but only slight differences in the metabolism of thiol reactants were found (not shown).
It is well known that some drugs (i.e. acetaminophen,
ethacrynic acid, and dapsone) (42-44) have different modes of action in rat and man, in terms of dose response curves and toxic limit concentrations; it is thereby possible that as these compounds are able
to react with SH groups, such differences can be due (at least in part)
to the presence of high reacting thiols on rat Hb. Because the rat is
widely used as a model to study the hemotoxicity of xenobiotics, we
consider that our results pose an important question and can help to
clarify some aspects of metabolism of thiol reactants by rat and human blood.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Neuroscience, Pharmacology Section, University of Siena, Via A. Moro 4, 53100 Siena, Italy. Tel.: 390577-234097; Fax: 390577-234098; E-mail: DISIMPLICIO@UNISI.IT.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M005156200
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ABBREVIATIONS |
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The abbreviations used are: PSH, protein sulfhydryl; BCNU, N,N'-bis-(2-chloroethyl)-N-nitrosourea; CDNB 1-chloro-2, 4-dinitro-benzene; DTNB, 5,5-dithio-bis(2-nitrobenzoic acid); Cys-NO, S-nitrosocysteine; EA, ethacrinic acid; GSSP, glutathione-protein mixed disulfhyde; Hb-SH, hemoglobin sulphydryl; GR, glutathione reductase; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; Hct, hematocrit.
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REFERENCES |
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1. | Meister, A. (1989) in Glutathione, Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, R., and Avramovic, O., eds) Part A, pp. 1-49, John Wiley & Sons, New York |
2. | Smith, C. V., and Mitchell, J. R. (1989) in Glutathione, Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, R., and Avramovic, O., eds) Part B, pp. 1-43, John Wiley & Sons, New York |
3. | Torchinsky, Y. M. (1981) Sulphur in Proteins , Pergamon Press, Oxford |
4. | Ziegler, D. M. (1985) Annu. Rev. Biochem. 54, 305-320[CrossRef][Medline] [Order article via Infotrieve] |
5. | Cotgreave, I. A., and Gerdes, R. G. (1998) Biochem. Biophys. Res. Commun. 242, 1-9[CrossRef][Medline] [Order article via Infotrieve] |
6. | Di Simplicio, P., Lusini, L., Giannerini, F., Giustarini, D., Bellelli, A., Boumis, G., Amiconi, G., and Rossi, R. (1998) in Nitric Oxide and the Cell: Proliferation and Death (Moncada, S. , Nisticò, G. , Bagetta, G. , and Higgs, E. A, eds) , pp. 47-59, Portland Press Ltd., London |
7. | Rossi, R., Lusini, L., Giannerini, F., Giustarini, D., Lungarella, G., and Di Simplicio, P. (1997) Anal. Biochem. 254, 215-220[CrossRef][Medline] [Order article via Infotrieve] |
8. | Gilbert, H. F. (1990) Adv. Enzymol. Relat. Areas. Mol. Biol. 63, 69-172[Medline] [Order article via Infotrieve] |
9. | Drodge, W., Eck, H. P., Mihm, S., and Galter, D. (1994) in Oxidative Stress, Cell Activation and Viral Infection (Pasquier, C. , Olivier, R. Y. , Auclair, C. , and Packer, L., eds) , pp. 285-299, Birkhauser Verlag, Basel |
10. | Jia, L., Bonaventura, C., Bonaventura, J., and Stamler, J. S. (1996) Nature 380, 221-226[CrossRef][Medline] [Order article via Infotrieve] |
11. | Stamler, J. S., Jia, L., Eu, J. P., McMahon, T. J., Demchenko, I. T., Bonaventura, J., Gernert, K., and Piantadosi, C. A. (1997) Science 276, 2035-2037 |
12. |
Rossi, R.,
Barra, D.,
Bellelli, A.,
Boumis, G.,
Canofeni, S.,
Di Simplicio, P.,
Lusini, L.,
Pascarella, S.,
and Amiconi, G.,.
(1998)
J. Biol. Chem.
273,
19198-19206 |
13. | Sies, H., and Ketterer, B. (1988) Glutathione Conjugation , Academic Press, San Diego, CA |
14. | Klotzsch, H., and Bergmeyer, H. U. (1965) in Methods in Enzymatic Analysis (Bergmeyer, H. U., ed) , pp. 363-366, Academic Press, New York |
15. | Rossi, R., Cardaioli, E., Scaloni, A., Amiconi, G., and Di Simplicio, P. (1995) Biochim. Biophys. Acta 1243, 230-238[Medline] [Order article via Infotrieve] |
16. | Condò, S. G., Giardina, B., Barra, D., Gill, S. J., and Brunori, M. (1981) Eur. J. Biochem. 116, 243-247[Abstract] |
17. | Riggs, A. (1981) Methods Enzymol. 76, 5-29[Medline] [Order article via Infotrieve] |
18. | Giardina, B., and Amiconi, G. (1981) Methods Enzymol. 76, 417-427[Medline] [Order article via Infotrieve] |
19. | Butler, E. (1969) J. Clin. Invest. 48, 1957-1966[Medline] [Order article via Infotrieve] |
20. | Saville, B. (1958) Analyst 83, 670-672 |
21. | Ross, D., Thor, H., Orrenius, S., and Moldeus, P. (1985) Chem. Biol. Interact. 55, 177-184[Medline] [Order article via Infotrieve] |
22. | Awasthi, S., Singhal, S. S., Srivastava, S. K., Torman, R. T., Zimniak, P., Bandorowicz-Pikula, J., Singh, S. V., Piper, J. T., Awasthi, Y, C., and Pikula, S. (1998) Biochemistry. 37, 5231-5238[CrossRef][Medline] [Order article via Infotrieve] |
23. | Mannervik, B., and Danielson, U. H. (1988) CRC Crit. Rev. Biochem. 23, 283-337[Medline] [Order article via Infotrieve] |
24. | Mathews, W. R., and Kerr, S. W. (1993) J. Pharmacol. Exp. Ther. 267, 1529-1537[Abstract] |
25. | Stern, A. (1985) in Oxidative Stress (Sies, H., ed) , pp. 331-349, Academic Press, London |
26. | Winterbourn, C. C., French, J. K., and Claridge, R. F. C. (1979) Biochem. J. 179, 665-673[Medline] [Order article via Infotrieve] |
27. | Becker, K., and Schirmer, R. H. (1995) Methods Enzymol. 251, 173-188[Medline] [Order article via Infotrieve] |
28. | Garel, M. C., Beuzard, Y., Thillet, J., Domenget, C., Martin, J., Galacteros, F., and Rosa, J. (1982) Eur. J. Biochem. 123, 513-519[Abstract] |
29. | Meister, A. (1983) Biochem. Soc. Trans. 11, 793-794[Medline] [Order article via Infotrieve] |
30. | Jocelyn, P. C. (1972) Biochemistry of the SH Group , Academic Press, London |
31. | Halasz, P., and Polgar, L. (1976) Eur. J. Biochem. 71, 571-575[Abstract] |
32. | Brodie, A. E., and Reed, D. J. (1987) Biochem. Biophys. Res. Commun. 148, 120-125[Medline] [Order article via Infotrieve] |
33. | Ferranti, P., Carbone, V., Sannolo, N., Fiume, I., and Malorni, A. (1993) Int. J. Biochem. 25, 1943-1950[Medline] [Order article via Infotrieve] |
34. | Di Simplicio, P., Cacace, M. G., Lusini, L., Giannerini, F., Giustarini, D., and Rossi, R. (1998) Arch. Biochem. Biophys. 355, 145-152[CrossRef][Medline] [Order article via Infotrieve] |
35. | Di Simplicio, P., and Rossi, R. (1994) Biochim. Biophys. Acta 1199, 245-252[Medline] [Order article via Infotrieve] |
36. | Di Simplicio, P., Lupis, E., and Rossi, R. (1996) Biochim. Biophys. Acta 1289, 252-260[Medline] [Order article via Infotrieve] |
37. | Mannervik, B. (1986) in Thioredoxin and Glutaredoxin Systems: Structure and Function (Holmgren, A. , Brandén, C. I. , Jornvall, H. , and Sjoberg, B. M., eds) , pp. 349-356, Raven Press, New York |
38. | Wefers, H., and Sies, H. (1983) Arch. Biochem. Biophys. 224, 568-578[Medline] [Order article via Infotrieve] |
39. | Wheeler, G. P., Johnston, T. P., Bowdon, B. J., McCaleb, G. S., Hill, D. L., and Montgomery, J. A. (1977) Biochem. Pharmacol. 26, 2331-2236[Medline] [Order article via Infotrieve] |
40. | Frischer, H., and Ahmad, T. (1977) J. Lab. Clin. Med. 89, 1080-1086[Medline] [Order article via Infotrieve] |
41. | Kelm, M., and Schrader, J. (1990) Circ. Res. 66, 1561-1575[Abstract] |
42. | Davis, C. D., Potter, W. Z., Jollow, D. J., and Mitchell, J. R. (1974) Life Sci. 14, 2099-2109[CrossRef][Medline] [Order article via Infotrieve] |
43. | Zins, G. R., Walk, R. A., Gussin, R. Z., and Ross, C. R. (1968) J. Pharmacol. Exp. Ther. 163, 210-215[Medline] [Order article via Infotrieve] |
44. | Vage, C., Saab, N., Woster, P. M., and Svensson, C. K. (1994) Toxicol. Appl. Pharmacol. 129, 309-316[CrossRef][Medline] [Order article via Infotrieve] |