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Blood, 1 September 2000, Vol. 96, No. 5, pp. 1985-1988
RED CELLS
Red cells from glutathione peroxidase-1-deficient mice have
nearly normal defenses against exogenous peroxides
Robert M. Johnson,
Gerard Goyette Jr,
Yaddanapudi Ravindranath, and
Ye-Shih Ho
From the Department of Biochemistry and Molecular
Biology, the Department of Pediatrics, and the Institute of Chemical
Toxicology, Wayne State Medical School, Detroit, MI.
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Abstract |
The role of glutathione peroxidase in red cell anti-oxidant defense
was examined using erythrocytes from mice with a genetically engineered
disruption of the glutathione peroxidase-1 (GSHPx-1) gene. Because
GSHPx-1 is the sole glutathione peroxidase in the erythrocyte, all red
cell GSH peroxidase activity was eliminated. Oxidation of hemoglobin
and membrane lipids, using the cis-parinaric acid
assay, was determined during oxidant challenge from cumene hydroperoxide and H2O2. No difference was
detected between wild-type red cells and GSHPx-1-deficient cells, even
at high H2O2 exposures. Thus, GSHPx-1 appears
to play little or no role in the defense of the erythrocyte against
exposure to peroxide. Simultaneous exposure to an
H2O2 flux and the catalase inhibitor
3-amino-1,2,4-triazole supported this conclusion. Hemoglobin oxidation
occurred only when catalase was depleted. Circulating erythrocytes from
the GSHPx-1-deficient mice exhibited a slight reduction in membrane thiols, indicating that high exposure to peroxides might occur naturally in the circulation.
(Blood. 2000;96:1985-1988)
© 2000 by The American Society of Hematology.
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Introduction |
The role of glutathione peroxidase (GSHPx) in
cellular defense against oxidant attack has been discussed for many
years.1-13 The red cell has been a central focus of this
research,1,2,4-6,8,9,11,12,14 because it is thought to
undergo a high endogenous rate of H2O2 production from hemoglobin autoxidation,15-21 which can be
markedly increased in cells with unstable
hemoglobins.22-24 In addition, the red cell is probably
exposed to H2O2 from stimulated macrophages and, under certain circumstances, from pathogenic bacteria or malarial
parasites.6,25 It is believed that the 2 enzymes, catalase
and glutathione peroxidase, protect the erythrocyte against peroxides
that are generated intracellularly or exogenously. The relative
importance of catalase and GSHPx in defending the cell has been debated
since the discovery of glutathione peroxidase.1 Arguments
have been presented for a predominant role for GSHPx1,2,18 or for catalase4,8,9,11,12 or for both playing a role in
detoxifying peroxide.3,5,6 The existence of naturally occurring acatalasemia in humans and mice has aided in resolving this
question, allowing the observation that acatalasemic red cells have
enhanced sensitivity to exogenous peroxides.25-27 However, a definitive solution to the question has been hindered by the absence
of an effective inhibitor of GSHPx, so that the effect of eliminating
GSHPx activity cannot be determined. Hochstein et al2,18
attempted to circumvent this problem by eliminating GSH, the obligatory
substrate of GSHPx, from the red cell. However, this approach suffers
from relative nonspecificity because GSH may have additional roles
within the red cell.28
The production of a mouse in which the GSHPx-1 gene has been
disrupted29 allows a definitive answer to the question of
GSHP function within the red cell. There are 4 distinct GSHPx
enzymes,13 but GSHPx-1 appears to be the only one to occur
in the erythrocyte. Thus, deletion of the gene for GSHPx-1 eliminates
all GSHPx activity in the red cell. We here describe the response of
GSHPx-1-negative erythrocytes to exogenous peroxides. Our results
suggest that GSHPx has a role in anti-oxidant defense, but only in
situations of high peroxide flux.
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Materials and methods |
Erythrocyte assays
For these experiments, blood was obtained from the hearts of
GSHPx-1-deficient mice and matched controls after Nembutol
(Abbott Laboratories) was administered for anesthesia. White cells were removed by filtration through cellulose,30 and the
erythrocytes were washed twice in phosphate-buffered saline (PBS) (145 mmol/L NaCl, 5 mmol/L NaPi, 1 mmol/L EDTA, pH 7.4). GSH and membrane thiols were determined as described earlier.31 For
glutathione peroxidase activity, cellulose-filtered cells were washed
once in PBS. Packed cells were diluted 1:20 in 0.01%
 -mercaptoethanol and 1 mmol/L EDTA, pH 7.4, and were flash frozen in
dry ice-methanol to lyse the cells. These hemolysates were assayed for
glutathione peroxidase as described by Beutler,32 with one
modification. To eliminate interference from
methemoglobin,33 10 mg NaCN and 30 mg
K3Fe(CN)6 was added to 10 mL of the 1 mol/L
Tris, 5 mmol/L EDTA, pH 8.0 buffer used in the assay.
Catalase was assayed as described by Aebi.34 To estimate
the catalase concentration, it was assumed that the specific activity of mouse catalase is similar to that of human, and a rate constant of
3.4 × 107 (mol/L) 1 s1 was
used. H2O2 was assayed by the method of Green
and Hill.35 The concentration of
H2O2 in the stock solution was
quantitated36 using an A240 value of 43.6 (mol/L)1 cm1. Methemoglobin (metHb) was
determined by a standard method37 or
spectroscopically.15 Unless otherwise noted, reagents were obtained from Sigma.
Knockout mice
The construction of the knockout mice has been
described29 and will be briefly outlined. The mouse genomic
clone for GSHPx-1 was isolated from a genomic library of the 129/SVJ
mouse. A 5.3-kb genomic fragment was selected and subcloned into
pBluescript SK for mapping and sequencing. The GSHPx-1 gene has 2 exons, and the second was disrupted by insertion of a Neo cassette. The
HSV thymidine kinase gene was inserted downstream of the GSHPx-1 gene to allow positive-negative selection. The vector was transfected into
R1 embryonic stem cells. Cells resistant to both ganciclovir and G418
were screened by Southern analysis with a sequence 3' to the targeted
sequence. Chimeric mice were obtained by injecting homologous
recombinants of R1 cells into C57BL/6 blastocysts. Homozygous
GSHPx-1-deficient mice were obtained by breeding heterozygous GSHPx(±) mice.
The absence of GSHPx activity was verified
enzymatically.32,33 The GSHPx-1-deficient red cells had
0.9 IU glutathione peroxidase/gHb, whereas wild-type cells had 310 IU/gHb. For comparison, human red cells had 26 IU/gHb.
Parinaric acid assay for lipid oxidation
The procedures of van den Berg et al38 were followed
exactly. The washed erythrocytes were adjusted to exactly 0.10%
hematocrit in PBS containing 10 mmol/L glucose. A cuvette with 2 mL of
this suspension and a small stir bar were placed in a
spectrofluorometer (model 8000; SLM/Aminco Instruments). The excitation
wavelength was 320 nm (slit width, 5 nm), and the emission wavelength
was 415 nm (slit width, 20 nm). Temperature was 25°C. Readings of fluorescence were begun, and at 30 seconds, 2 µL 1 mmol/L
cis-parinaric acid (Molecular Probes, Eugene, OR) was added
to make the final concentration 1 µmol/L. At 150 seconds, appropriate
volumes of 1 mmol/L cumene hydroperoxide (Aldrich Chemicals, St Louis,
MO) dissolved in ethanol were added. Fluorescence was recorded for 10 minutes. Controls were treated in the same way, except that neat
ethanol replaced the cumene hydroperoxide solution. As noted earlier,38 there was a gradual decline in fluorescence
even in the control because of the high light emission of the
SLM instrument.
Hemoglobin oxidation in response to exogenous
H2O2
A continuous flux of H2O2 was generated
with glucose oxidase.5,8,9,12,39,40 Washed,
cellulose-filtered red cells were resuspended to 20% hematocrit in
Krebs-Ringer buffer (143 mmol/L NaCl, 5.7 mmol/L KCl, 1.4 mmol/L
MgCl2, 18 mmol/L NaPi, pH 7.4) with 10 mmol/L glucose and
50 µg/mL gentamicin. To begin the exposure to
H2O2, glucose oxidase was added. The rate of
H2O2 production under these conditions was
linear with added glucose oxidase and was equal to 0.123 µmol/L of
H2O2 produced per minute for each mU glucose
oxidase/mL. Both glucose concentration and pH were monitored, and did
not change during the incubation.
| |
Results |
Erythrocyte oxidation in response to organic peroxides
Addition of cumene hydroperoxide to lipid bilayers leads to the
oxidation of membrane-lipid components. This can be conveniently assayed by monitoring the disappearance of the fluorescent signal of
cis-parinaric acid intercalcated into the
bilayer.41 Van den Berg et al38 have carefully
optimized the cis-paranaric acid assay for use with intact
red cells. Using their procedures, we detected no difference between
GSHPx-1-deficient and wild-type red cells in their responses to cumene
hydroperoxide challenge (Figure 1),
indicating that GSHPx-1 plays no role in protecting red cell membrane
lipids from oxidant attack. In addition, analysis of the hemoglobin in
these cells demonstrated that metHb formation in response to cumene
hydroperoxide was negligible in both types of erythrocytes.
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| Figure 1.
Fluorescence intensity of 50 µmol/L cis-parinaric acid
in a 2 mL suspension of red cells, 10% hematocrit, in PBS with 10 mmol/L glucose and 50 µg/mL gentamycin.
Readings were taken every 15 seconds. After 150 seconds of
equilibration, either 50 µL cumene hydroperoxide in ethanol was added
(100 µmol/L final concentration) (filled symbols), or 50 µL ethanol
(open symbols) was added to controls. There was no difference between
the rate of fluorescence loss in wild-type red cells (circles) and
GSHPx-1-deficient red cells (squares).Two runs are shown.
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Hemoglobin oxidation in response to
H2O2
Single additions of H2O2 to red cell
suspensions were rapidly destroyed. This was true for both wild-type
and GSHPx-1-deficient erythrocytes. Therefore, a glucose
oxidase-glucose system was used to produce a steady flux of
H2O2 in the test solution.
H2O2 fluxes were varied over a 50-fold range
from 0.12 µmol/L/min to 6.15 µmol/L/min (Table
1). Despite the high rates of H2O2 generation, steady state
H2O2 levels in the supernatant were undetectably low when red cells were present. In most experiments, the
rate of hemoglobin oxidation was negligible, though some variability in
oxidation rates was noted. Significantly, however, no difference was
detected in the rate of hemoglobin oxidation between wild-type and
GSHPx-1-deficient cells in these experiments, indicating that GSHPx
plays no role in protecting erythrocyte hemoglobin against oxidation by
peroxides.
Relative importance of catalase and GSHPx
This result implied that GSHPx was unnecessary to protect red
cells against exogenous H2O2 fluxes, a
conclusion that was supported by experiments with the catalase
inhibitor, 3-amino-1,2,4-triazole (3-AT). The compound irreversibly
inhibits catalase, but only when catalase is actively reducing
H2O2. Catalysis by catalase proceeds in
2 steps:
Catalase (ground state) + H2O2  Compound
I + H2O
Compound I + H2O2 Catalase + H2O + O2
Margoliash42 found that 3-AT irreversibly combines with
Compound I, which is the product of the reaction between catalase and
the first molecule of H2O2. Thus, loss of
catalase activity in the presence of 3-AT is an indication that
catalase is actively catabolizing H2O2.
As has been found with erythrocytes from other species, exposure of
wild-type mouse red cells to a continuous flux of
H2O2 in the presence of 3-AT led to a slow
inactivation of catalase (Figure 2A).
When H2O2 fluxes were low to moderate, the rate
of catalase inactivation was similar in both wild-type and
GSHPx-deficient erythrocytes. At high H2O2 flux
rates, however, catalase was more rapidly inactivated in
GSHPx-deficient red cells than in controls. Thus, catalase is
sufficient to deal with low to moderate H2O2 fluxes because there is no effect of GSHPx-1 deletion. At high fluxes,
however, the more rapid rate of catalase inactivation in
GSHPx-deficient cells than in the wild type strongly implies that
GSHPx-1 assists in eliminating H2O2 under these
circumstances. The data are consistent with a model in which wild-type
cells exposed to high H2O2 levels use both
catalase and GSHPx for H2O2 removal. In
GSHPx-deficient cells, the entire exogenous
H2O2 flux will combine with catalase, thus
raising the level of Compound I, leading to a more rapid inactivation
by 3-AT.
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| Figure 2.
Catalase inactivation (A) and hemoglobin oxidation (B)
in red cells exposed to 50 mmol/L 3-AT and a continuous flux of
H2O2.
Hematocrit 10%, Krebs-Ringer buffer. The rate of
H2O2 production was 0.123µmol/L per minute
(circles), 0.615 µmol/L per minute (squares), and 1.23 µmol/L per
minute (triangles). Solid lines, wild-type red cells; dashed lines,
GSHPx-1-deficient red cells.
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The importance of catalase is also indicated by the observation that
metHb rose only after catalase was inactivated (Figure 2B). Hemoglobin
oxidation was seen when approximately 50% of the catalase was
inactivated (Figure 3).
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| Figure 3.
Hemoglobin oxidation as a function of catalase
inactivation.
The percentage methemoglobin in GSHPx-1-deficient cells is plotted
versus the residual catalase activity (percentage of starting
activity).
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Membrane thiols
Table 2 shows that the levels of
endogenous GSH and metHb were normal in GSHPx-1-deficient red cells.
However, there was a statistically significant decrease in membrane
protein thiols in red cells obtained from GSHPx-1-deficient mice
(P < .001). Each table entry shows values for the pooled
blood from 2 to 3 mice.
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Discussion |
The defenses of the red cell against oxidant attack include
catalase and glutathione peroxidase. There has been a long debate about
the relative importance of these 2 enzymes.1-6,8,9,11,12,18,43 Catalase, the first
discovered, was thought to play a major role in protecting the cell,
but the discovery of glutathione peroxidase and experiments
demonstrating an essential role for glutathione in protecting
intracellular hemoglobin from oxidation led to the proposal that GSHPx
was the major defensive enzyme.1,2,18,44 However,
considerations of kinetic constants4,9,11,12 and reconstitution studies with resealed ghosts14,43 are more
consistent with a predominant role for catalase.
The observations presented here with red cells lacking GSHPx-1 indicate
that under most conditions, GSHPx-1 is a dispensable enzyme. No
oxidation of hemoglobin or membrane lipid was observed when
GSHPx-deficient red cells were exposed to exogenous peroxides. A
difference between wild-type and GSHPx-deficient erythrocytes was
detected only in the presence of 3-AT and high exogenous
H2O2 exposures. Thus, GSHPx-1 in the red cell
has a functional role only under conditions of severe oxidant stress.
This finding confirms the earlier inferences of Scott et
al,8 who used resealed ghosts containing mixtures of
catalase and GSHPx, and it confirms the conclusions of Gaetani et
al11 and Mueller et al,12 based on the
kinetics of mixtures of purified catalase and GSHPx. All these groups
modeled low to moderate levels of oxidant stress and concluded that
GSHPx has little role in the erythrocyte's oxidant defense. Our
results with the GSHPx-1-deficient mouse verify their models,
providing direct evidence that GSHPx plays little or no role in red
cell anti-oxidant defense, except possibly when oxidant exposures are high.
It is difficult to ascertain whether the in vivo levels of oxidant
exposure are ever high enough to bring GSHPx into play. The results
depicted in Table 2, showing that the circulating red cells of
GSHPx-deficient mice exhibit some degree of membrane protein oxidation,
suggests that oxidant exposures in the circulation may sometimes be
high enough to damage GSHPx-deficient red cells. It has been
speculated6,25 that peroxides and nitric oxide produced by
macrophages and endothelial cells or by pathogenic bacteria or
parasites can generate localized high concentrations of
H2O2, which might explain the observed
endogenous red cell membrane oxidation. However, this inference does
not affect the main conclusion of this work, which is that red cell
GSHPx-1 plays little or no role in erythrocyte anti-oxidant defenses,
at least in the mouse. Although absolute concentrations
differ,7 the array of antioxidant enzymes is similar in
mouse and other mammals, including humans, suggesting that this result
will be of general applicability. In support of this idea, it can be
noted that sporadic cases of GSHPx deficiency have been noted in humans
without any accompanying clinical symptoms.45 Thus,
genetic, enzymologic, and clinical evidence all suggest that GSHPx is
of minor significance for red cell function.
| |
Footnotes |
Submitted March 7, 2000; accepted May 1, 2000.
Supported by National Institutes of Health grant HL56421 (Y-S.H.) and
the Ginopolis Fund of Children's Hospital of Michigan.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Robert M. Johnson, Department of Biochemistry and
Molecular Biology, Wayne State Medical School, 540 E. Canfield,
Detroit, MI 48201; e-mail: rmjohns{at}med.wayne.edu.
| |
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Abstract
Introduction