Available online at www.sciencedirect.

com

Toxicology in Vitro 21 (2007) 1355–1364

www.elsevier.com/locate/toxinvit

Review

In vitro models of oxidative stress in rat erythrocytes:

Effect of antioxidant supplements
C.S. Shiva Shankar Reddy a, M.V.V. Subramanyam b, R. Vani a, S. Asha Devi a,*

a
Laboratory of Gerontology, Department of Zoology, Bangalore University, Bangalore 560 056, India
b
Department of Sericulture, Bangalore University, Bangalore 560 056, India

Received 3 March 2007; accepted 22 June 2007

Available online 30 June 2007

Abstract

The present study was designed to induce oxidative stress in lipid and aqueous phases through azo bis(2-amidinopropane)dihydro-
chloride (AAPH), 2,2 0 -azobis 2,4-dimethylvaleronitrile (ADVN) and hydrogen peroxide (H2O2) either alone or in combination with vita-
min C or vitamin El and to assess the vulnerability of rat erythrocytes to oxidative stress. While AAPH acted equally on cell membrane
and cytosol, ADVN increased OS in the membrane. The extent of hemolysis and increased membrane fragility caused was more in the
case of azo compounds than of H2O2. While vitamin E (2 mM) reduced oxidative stress in the membrane, vitamin C (60 mM) was more
effective in the lysates. The concentration of malondialdehyde and advanced oxidation protein products was lowered by antioxidants.
The level of lipofuscin, a product of lipid peroxidation was also increased by ADVN and H2O2. Antioxidants, did, however, reduce
the accumulation of protein carbonyl content in cells exposed to azo compounds although they were ineffective in inhibiting oxidation
of membrane band 3 protein and sulphydryl content. Taken together, our study demonstrated the antioxidative property of vitamin E
and vitamin C in reducing oxidative stress in aqueous as well as lipid phases of erythrocytes and further suggested the feasibility of
in vitro models in evaluating the mechanisms of oxidative injury.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Antioxidants; Azo compounds; Band 3; Erythrocytes; Hemolysis; Osmotic fragility; Protein oxidation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1356
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1356
2.1. Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1356
2.2. Blood sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1356
3. Induction of OS by aqueous and nonaqueous compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1356
4. Preparation of erythrocyte ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1357
5. Oxidative stress indices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1357
5.1. Hemolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1357
5.2. Osmotic fragility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1357

Abbreviations: AOPP, advanced oxidation protein products; AAPH, 2,2 0 -azobis(2-amidinopropane dihydrochloride; ADVN, 2,2 0 -azobis(2,2-di-
methylvaleronitrile; AMVN, 2,2 0 -azobis(4-methoxy-2,4-dimethylvaleronitrile); CAT, catalase; FRs, free radicals LPO lipid peroxidation; LF, lipofuscin;
MDA, malondialdehyde; OS, oxidative stress; PUFA, polyunsaturated fatty acids; PrC, protein carbonyl; P-SH, protein sulphydryl; ROS, reactive oxygen
species.
*
Corresponding author. Tel.: +91 080 2296 1562.
E-mail address: asuba@blr.vsnl.net.in (S. Asha Devi).

0887-2333/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2007.06.010
1356 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364

5.3. Lipid peroxidation (LPO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ . . 1357

5.4. Lipofuscin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ . . 1357
5.5. Protein oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ . . 1357
5.6. Membrane protein sulfhydryl group . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ . . 1357
5.7. Electrophoresis of membrane protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ . . 1358
5.8. Protein determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ . . 1358
6. Statistical analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ . . 1358
7. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... . . 1358
8. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... . . 1362
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... . . 1363
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... . . 1363

1. Introduction (4) to test the influence of single antioxidants, vitamin C

Oxidative stress plays an important role in many physi-
ological and pathological phenomena. Erythrocytes have
been used as a model to investigate oxidative damage in
biomembranes because of their high vulnerability to perox-
idation. Exposure of erythrocytes to OS under conditions
such as exercise (Asha et al., 2005) and hypoxia lead to
lipid peroxidation, changes in cellular morphology, protein
cross-linking, hemolysis and conformation of membrane
proteins (Okamoto et al., 2004; Fujino et al., 2000).
Although erythrocytes contain an extensive antioxidant
defense system, oxidative damage of membrane proteins
and lipids contribute to the senescence of normal cells that
result in a shorter life span for damaged cells. The major
source of intracellular reactive oxygen species in the eryth-
rocyte is autoxidation of oxyhemoglobin, which generates
superoxide, and through dismutation produces hydrogen
peroxide (Nagababu et al., 2003). Earlier, we have demon-
strated that the oxidative stress experienced by erythrocytes
in vivo under the intermittent hypobaric hypoxic conditions
can be suppressed by intervention of dietary antioxidants
(Asha et al., 2007).
The decomposition of a water-soluble azo compound,
2,2,-azobis(2-amidinopropane hydrochloride, AAPH) at
physiological temperature generates free radicals in vitro
that can attack the erythrocyte membrane and induce lipid
peroxidation (LPO) leading to hemolysis (Liu et al., 2002).
Hydrogen peroxide is considered a key oxygen-free radical
because of its relatively high stability, diffusion, and
involvement in cell signaling cascades.
In this paper, we report on two in vitro models, aqueous
and lipid phase models of oxidative stress using free radical
initiators such as AAPH and H2O2 (water-soluble), and
ADVN (lipid-soluble) in erythrocytes. Since the rate of free
radical generation from these compounds can be easily
controlled in vitro, we have used them to examine the oxi-
dant-induced stress markers.
The objectives of the present study were: (1) to induce oxi-
dative stress by AAPH, ADVN and H2O2 in intact erythro-
cytes, (2) to analyze the markers of oxidative stress, namely
hemolysis, osmotic fragility and lipid peroxidation, (3) to
investigate the effects on protein oxidation products, and
and vitamin E in protecting the cells against oxidative
stress.
2. Materials and methods
2.1. Chemicals
Hemoglobin reagent was obtained from Coral Clinical
Systems, Goa, India. 2,2 0 -azobis (2-amidinopropane) dihy-
drochloride (AAPH) and 2,2 0 -azobis(2,4-dimethylvaleronit-
rile) (ADVN) was purchased from Wako Pure Chemical
Industries Ltd (Osaka, Japan). Acrylamide, bis, thiobarbi-
turic acid (TBA), sodium dodecylsulphate (SDS), 1,1,3,3-
tetramethoxy propane (TMP), 5,5 0 -dithio-bis 2-itrobenzoic
acid (DTNB) and bovine serum albumin (BSA) stock were
purchased from Sigma–Aldrich Chemicals (St. Louis, MO,
USA). Molecular markers for protein were obtained from
Bangalore Genei (Bangalore, India). All other chemicals
used were of reagent grade and organic solvents were of
spectral grade.
2.2. Blood sampling
Blood sampling was similar to our earlier method (Asha
et al., 2005). In brief, blood was carefully aspirated from the
heart into 10% ethylenediaminetetracetic acid (EDTA)-
coated tubes.
Erythrocytes were isolated by centrifugation for 20 min
at 1000g, and at 4 C. The plasma and buffy coat were
removed by aspiration. The cells were washed three times
with 310 mOsm isotonic phosphate buffer (pH 7.4), and
finally suspended in an equal volume of isotonic phosphate
buffer. This constituted the erythrocyte suspension (Dodge
et al., 1963).
3. Induction of OS by aqueous and nonaqueous compounds
Oxidative stress was induced in 500 ll whole cell suspen-
sion by compounds that were either hydrophilic [AAPH or
 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364
H2O2] or lipophilic [ADVN]. AAPH generated peroxyl
radicals outside the membrane, a homogenous phase
whereas ADVN generated oxidative stress on the mem-
brane, a non-homogenous system (lipid phase). H2O2 that
is normally generated in vivo mainly by the autoxidation of
hemoglobin and dismutation of superoxide gave rise to
radicals like hydroxyl ions. Our incubation system con-
tained AAPH, a diazo compound of two different concen-
trations (10 and 50 mM) and H2O2 (0.5, 2 and 8 mM) in
isotonic buffer at pH 7.4, and a lipophilic diazo compound,
ADVN (10 and 50 mM) in isotonic buffer at pH 7.4. Eryth-
rocytes were exposed to the free radical initiators for
60 min.
The above procedure was repeated in a second set of
cells that were pretreated with antioxidants, vitamin C
(60 mM) or vitamin E (2 mM) for 30 min prior to exposure
to either the azo compounds or hydrogen peroxide.
4. Preparation of erythrocyte ghosts
Hemolysis was performed by pipetting 2 ml aliquots of
washed erythrocyte suspension in a tube containing 28 ml
of hypotonic buffer (20 mOsm, pH 7.4). The contents were
mixed by gentle swirling and then centrifuged at 20,000g
for 40 min. The supernatant was decanted carefully and
the ghost (membrane) was resuspended by adding same
strength buffer to reconstitute the original volume. The
ghosts were washed three times subsequent to hemolysis.
The membrane pellet was resuspended in the isotonic buffer
for the assay (Dodge et al., 1963).
5. Oxidative stress indices
5.1. Hemolysis
A 5% (v/v) suspension of washed, packed erythrocytes
in buffer (310 mOsm) was mixed with the same volume of
0.5, 2 and 8 mM H2O2. The mixtures were incubated at
37 C for 2 h. One sample of each concentration was pre-
treated with 1 mM sodium azide, an inhibitor of CAT, at
37 C for 10 min before inducing oxidative stress as above.
Hemolysis was determined by measuring released hemoglo-
bin into the supernatant of the induced samples in a spec-
trophotometer at 540 nm and was represented on the basis
of the maximum absorbance (100%) in the aliquots of
erythrocytes completely hemolysed in distilled water (Sen-
turk et al., 2001).
5.2. Osmotic fragility
The procedure was a slightly modified method of O’Dell
et al. (1987). A 100 ll aliquot of washed erythrocyte sus-
pension was added to tubes containing 0.3%, 0.4%, and
0.9% buffered salt solution (BSS, pH 7.4). These tubes were
allowed to stand at room temperature for 30 min, centri-
fuged at 1270 g to pellet the cells and the absorbance of
the supernatant was measured at 540 nm. Hemolysis in
1357
each tube was expressed as a percentage taking as 100%,
the maximum value of absorbance of the distilled water.
BSS (0.9%) was considered as a control sample.
5.3. Lipid peroxidation (LPO)
Malondialdehyde, a product of lipid peroxidation was
determined according to the method of Ohkawa et al.
(1979). In brief, 100 ll of lysate/membrane sample was
added to 8.1% SDS, vortexed and incubated at room tem-
perature. This was followed by the addition of 20% acetic
acid and 0.6% TBA, and placed in a boiling water bath
for 60 min. The samples were cooled and butanol: pyridine
was added and centrifuged at 640g. Absorbance of the col-
ored layer was measured at 532 nm with TMP as a stan-
dard. Malondialdehyde concentration was expressed
either as nmol/mg protein for the membranes or nmol/g
Hb for whole erythrocytes.
5.4. Lipofuscin
The samples were examined under a fluorescence micro-
scope (Olympus IX 70). In the micrographs lipofuscin pig-
ments appeared bluish in color. The cells were imaged
using a cool-snap PC-controlled camera coupled with
micro image software for the quantification of fluores-
cence, which was expressed in arbitrary units. An individ-
ual experiment (n) was observed in a field of 13–15 cells
on a cover slip. Experiments were repeated on five animals
on separate cover slips and results reported as mean ± SE.
5.5. Protein oxidation
Protein carbonyl content was measured as an index of
protein oxidation as described by Uchida and Stadtman
(1993). In brief, the experimental tube included 0.8 ml of
the lysate /membrane sample in isotonic buffer with an
equal volume of 0.1% (w/v) 2,4-DNPH in 2N HCl, and
the control tube contained equal volume of the sample
and 2N HCl. Both the sets were incubated at room temper-
ature for 60 min. After the incubation, 20% trichloroacetic
acid was added and the contents centrifuged at 1900g.
After washing with ethanol:ethylacetate mixture (50:50),
the residue was dissolved in 8 M guanidine hydrochloride
in 133 mM Tris solution (pH 7.2) containing 13 mM
EDTA and centrifuged at 1900g. The absorbency of each
sample was read at 365 nm in an UV/VIS spectrophotom-
eter (ELICO, Model SL 159) against the control. The
results were expressed as lmoles of 2,4-DNPH-incorpo-
rated/mg protein based on a molar extinction coefficient
of 2.1 · 104 M1 cm1 for aliphatic hydrazones.
5.6. Membrane protein sulfhydryl group
The concentration of P-SH groups in the membrane
proteins was measured as described by Habeeb (1972).
In brief, 1.5 ml of 0.08 mol/L sodium phosphate buffer
1358 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364
(pH 8.0) containing Na2-EDTA, and SDS was added to
each assay tube containing 120 lg of membrane protein.
DTNB was added and the solution was vortexed. Color
was allowed to develop for 15 min at room temperature
and absorbance was measured at 412 nm, using an equiva-
lent concentration of protein as the blank. Sulphydryl con-
centration was calculated from the net absorbance and
molar absorptivity, 13,600 mol/L1 cm1.
5.7. Electrophoresis of membrane protein
SDS–polyacrylamide gel electrophoresis (SDS–PAGE)
of the membrane was performed by Laemmli’s method
(1970). The prepared membrane was solubilized to a final
concentration of 100 lg (protein)/ml in Laemmli sample
buffer solution containing 0.0625 M Tris–HCl (pH 6.8),
2% SDS, 10% glycerol and 5% b-mercaptoethanol with
bromophenol blue. The solubilized membrane proteins
were loaded on to a 3.5% stacking gel and allowed to run
through a 12% separating gel. Electrophoresis was carried
out for 90 min and protein bands were visualized by stain-
ing with coomassie brilliant blue R-250 for an hour under
gentle shaking. The molecular weight of membrane pro-
tein, band 3 in the gel was estimated from the calibration
of standard proteins, ranging from 14 to 100 kDa. Band
3 was captured into a computer via an image scanner (Mul-
tiimageTM II light cabinet, DE-500) to analyze the area of
band using image analysis software (AlphaDigiDocTM
AD-1200 and 1201).
5.8. Protein determination
Protein was determined by the method of Lowry et al.
(1951) using BSA as the standard.
6. Statistical analyses
Results were represented as mean ± SE. Values between
the groups were analyzed by one-way ANOVA and were
considered significant at p < 0.05. Tukey–Kramer Multiple
Comparison Test was performed for lipid peroxidation and
protein oxidation. Two-way ANOVA was performed
between the groups and sub-groups to analyze hemolysis
and osmotic fragility followed by Bonferroni Post test,
using Graphpad Prism Software.
7. Results
Oxidative damage occurred in cells that were incubated
in all the three free radical initiators, ADVN, AAPH and
H2O2. The concentration of AAPH and ADVN ranged
from 10 mM to 50 mM, and of H2O2 ranged from 0.5 to
8 mM. The results of this study can be presented with
reference to four indices of oxidative stress, namely, hemo-
lysis, osmotic fragility, lipid peroxidation and protein
oxidation.
A 5-fold increase in AAPH and ADVN concentration
(10–50 mM) resulted in 104% and 93% hemolysis in eryth-
rocytes. The extent of increase was however low (27%) even
with a 16-fold increase in H2O2 (8 mM) (Table 1).
A second set of experiments were performed to relate the
observed hemolysis to changes in the osmotic fragility of
erythrocytes. Membrane fragility increased by 80% and
88% in 0.3 and 0.4% BSS respectively in cells exposed to
oxidative stress initiators and the increases were over the
respective controls. In 50 mM AAPH, increases by 162%
and 153% were seen in 0.3% and 0.4% BSS, respectively.
With a 5-fold increase in AAPH (50 mM), 45% and
34.3% increases in osmotic fragility were evident in 0.3%
and 0.4%, respectively, of BSS. Erythrocytes when exposed
to ADVN (10 mM) showed an increase in fragility by 120%
over the control in 0.3% as well as 0.4% BSS. With a 5-fold
increase in ADVN (50 mM), osmotic fragility remained
unaltered (Table 2a). Membrane fragility increased by
116% and 113% in 0.3% and 0.4% BSS, respectively, of cells
when exposed to H2O2 (Table 2b). In contrast to a 5-fold
increase in azo compounds, with a 16-fold increase in
H2O2 (8 mM), increases by 56% and 61% were noticeable
(Table 2b).
To establish whether there is a link between the altera-
tions seen in osmotic fragility and hemolysis and lipid per-
oxidation, we studied the two azo compounds in the
generation of malondialdehyde. While malondialdehyde
in the lysate increased by 540% and 739% in 10 mM and
50 mM AAPH respectively over the control, increases by
only 183% and 292% were noticeable in 10 mM and
50 mM ADVN respectively. With a 5-fold increase in
AAPH, a 30% elevation in malondialdehyde was seen,
whereas a similar extent of increase in ADVN resulted in
a 37% increase (Table 3a). MDA in the membrane increased
in 10 mM (56%) and 50 mM (68%)-AAPH exposed cells.
Treating the cells with ADVN (50 mM) induced a 45%
increase in malondialdehyde. While AAPH induced an
8.4% increase for a 5-fold increase in its concentration,
Table 1
(a) Azo compounds and (b) hydrogen peroxide – induced hemolysis
Groups AAPH ADVN
10 mM 50 mM 10 mM 50 mM
(a)
% Hemolysis 26.8 ± 5.1A 54.7 ± 4.8B 45.2 ± 4.0B 87.0 ± 3.8C
H2O2 concentration
0.5 mM 2 mM 8 mM
(b)
% Hemolysis 74.5 ± 0.5A 74.3 ± 2.2A 95.0 ± 1.9B
Hemolysis is expressed as %. Values are expressed as mean ± SE of five
animals/group AAPH [2,2 0 -azobis(2-amidinopropane)dihydrochloride]
and ADVN [2,2 0 -azobis(2,4-dimethylvaleronitrile)]. H2O2, hydrogen per-
oxide. Changes between the groups are analyzed by one-way ANOVA
followed by Tukey–Kramer multiple comparison test. ABC values with
different superscripts between groups in each column are significantly
different at p < 0.05.
 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364 1359

Table 2 Table 4
(a) Azo compounds and (b) hydrogen peroxide – induced osmotic fragility Effect of antioxidants on (a) azo compound and (b) H2O2 – induced lipid
peroxidation
Groups % NaCl Concentration
Groups Control AAPH AAPH + vit C AAPH + vit E
0.3a 0.4b 0.9c
(a)
(a)
Control 21.5 ± 1.2A 20.0 ± 1.3A 17.5 ± 1.2A MDA (L) 2.2 ± 0.2A 18.3 ± 0.9B 4.5 ± 0.1C 3.5 ± 0.1AC
MDA (M) 9.8 ± 0.5A 16.7 ± 0.6B 10.6 ± 0.1A 9.9 ± 0.4AC
10 mM AAPH 38.7 ± 3.7B 37.6 ± 4.4B 24.1 ± 2.3A
50 mM AAPH 56.5 ± 5.3CD 50.5 ± 4.0CD 36.1 ± 5.0B ADVN ADVN + vit C ADVN + vit E
10 mM ADVN 47.7 ± 0.8BD 45.3 ± 0.8BD 36.2 ± 1.5B
50 mM ADVN 48.0 ± 1.7BD 44.6 ± 1.4BD 34.4 ± 2.3B
MDA (L) 2.2 ± 0.2A 8.5 ± 1.1B 3.1 ± 0.1A 3.4 ± 0.1A
(b) MDA (M) 9.8 ± 0.5A 14.3 ± 0.6B 10.3 ± 0.2A 10.7 ± 0.2A
Control 15.5 ± 2.0A 15.2 ± 2.4A 12.6 ± 2.0A
H2O2 H2O2 + vit C H2O2 + vit E
0.5 mM 21.5 ± 1.4AC 20.0 ± 1.0A 17.8 ± 1.0A
2 mM 29.2 ± 1.4BC 23.3 ± 0.7A 20.8 ± 0.6AC
8 mM 33.6 ± 4.6B 32.3 ± 4.0B 29.0 ± 3.9BC (b)
MDA (L) 3.0 ± 0.3A 7.5 ± 0.1B 4.2 ± 0.1C 3.7 ± 0.1C
Hemolysis is expressed as %. Values are expressed as mean ± SE of five
MDA (M) 5.0 ± 0.2A 7.5 ± 0.1B 6.9 ± 0.1C 6.6 ± 0.1C
animals/group. AAPH [2,2 0 -azobis(2-amidinopropane)dihydrochloride]
and ADVN [2,2 0 -azobis(2,4-dimethylvaleronitrile)]. Changes between the MDA, Malondialdehyde; L, lysate; M, membrane. Values were expressed
groups are analyzed by two-way ANOVA followed by Bonferroni post hoc as mean ± SE of five animals/group. Cells were incubated individually
test. Values with different superscripts between groups in each row are with AAPH/ADVN [50 mM] /H2O2 [2 mM] and vit C (60 mM); vit E
significantly different at p < 0.05 are represented in upper case (ABCD). (2 mM). Changes between the groups are analyzed by one-way ANOVA
Changes within the group are represented in lower case (abc). followed by Tukey–Kramer post hoc test. Values with different super-
scripts between groups in each column are significantly different at
p < 0.05 are represented in upper case (ABC).
Table 3
Comparison between lipid peroxidation and protein oxidation
malondialdehyde by 36.5% and 25% over AAPH and
AAPH Cona 10 mMb 50 mMc
ADVN (50 mM) treated cells, respectively. Decreases by
(a) 75% and 63.5% over the ADVN cells were seen in response
MDA (L) 2.2 ± 0.2A 13.9 ± 1.4A 18.3 ± 0.9A
MDA (M) 9.8 ± 0.6B 15.4 ± 2.9A 16.7 ± 0.6A
to vitamin C (Table 4). In contrast to the azo compounds,
PrC (L) 5.8 ± 0.6A 14.3 ± 0.7A 23.0 ± 0.1B H2O2 increased malondialdehyde in the lysate by 114%,
PrC (M) 5.6 ± 0.2A 24.6 ± 1.4B 30.2 ± 0.6C 151% and 183% in 0.5 mM, 2 mM and 8 mM respectively.
ADVN Cona 10 mMb 50 mMc
With a 16-fold increase in H2O2, malondialdehyde
MDA (L) 2.2 ± 0.2A 6.2 ± 0.4A 8.5 ± 1.1A increased by 32% (Table 3b). Malondialdehyde in the mem-
MDA (M) 9.8 ± 0.6B 11.4 ± 0.3B 14.3 ± 0.6B brane increased by 140% in response to 8 mM and the
PrC (L) 5.8 ± 0.6C 14.5 ± 0.3C 25.0 ± 0.2C extent of increase was by 72% for a 16-fold increase in
PrC (M) 5.6 ± 0.2C 28.8 ± 1.0D 37.1 ± 1.3D H2O2. Antioxidants were effective in reducing malondialde-
H2O2 Cona 0.5 mMb 2 mMc 8 mMd
hyde by 50% (vitamin E) and 28% (vitamin C) of H2O2
(8 mM)-treated cells. In contrast to the lysate, vitamin E
(b)
MDA (L) 3.0 ± 0.3A 6.4 ± 0.2A 7.5 ± 0.1A 8.5 ± 1.5A
and C reduced malondialdehyde to a lesser extent, i.e. by
MDA M) 5.0 ± 0.2AC 7.0 ± 0.1A 7.5 ± 0.1A 12.1 ± 1.6BC 12% and 15% respectively over H2O2 (8 mM)-treated cells
PrC (L) 5.8 ± 0.6BC 7.3 ± 0.7A 8.1 ± 0.1A 8.2 ± 0.3A (Table 4). In addition to lipofuscin was measured as another
PrC (M) 4.3 ± 0.3AC 6.0 ± 0.3A 7.8 ± 0.1A 10.2 ± 1.7AC marker of lipid peroxidation. Fluorescence microscopic
MDA, Malondialdehyde; PrC, protein carbonyl; L, lysate; M, membrane. observation of erythrocytes showed an increase in lipofus-
Values are expressed as mean ± SE of five animals/group. Changes cin in the cells incubated with 10 mM (67%) and 50 mM
between the groups are analyzed by two-way ANOVA followed by Bon- (98%) ADVN when compared to control. (Fig. 1a and b).
ferroni post hoc test. Values with different superscripts between groups in
each row are significantly different at p < 0.05 are represented in upper
While a 5-fold increase in AAPH concentration increased
case (ABC). Changes between concentrations are represented in lower case lipofuscin increased by 33%, the same extent of 18%
(abcd). increase in response to ADVN was noticed. Further, eryth-
rocytes treated with 8 mM H2O2 showed elevation in LF by
199% over the untreated ones (Fig. 1c). Antioxidants were
ADVN elevated malondialdehyde to 25% for a similar fold ineffective in reducing lipofuscin concentration in azo- as
increase (Table 3a). Experiments were conducted to test the well as H2O2-treated cells (Fig. 1a–f).
potential of vitamin E and C for reducing oxidative stress Protein carbonyl level was measured as an index of
induced by azo compounds. We pre-incubated the lysates protein oxidation in erythrocytes by azo compounds with
and membranes with these antioxidants before incubating and without the antioxidants. The effects of AAPH were
them with AAPH and ADVN. Vitamin E and C reduced seen in terms of a 145% (10 mM) and 295% (50 mM)
malondialdehyde by 81% and 75% and by 60% and 63.6% increase in carbonyl level of lysates over the controls. Car-
over cells exposed to 50 mM AAPH and ADVN, respec- bonyl concentration increased by 61% for a 5-fold increase
tively (Table 4). Vitamin E effective in reducing membrane in AAPH concentration. Increases by 149% and 329% were
1360 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364

a 1500 d
B
Fluorescence Units/cell A 1200

Fluorescence Units/cell
A
1000 A B
1000
A
800

600
500
400

200

0 0
Con 10mM 50mM Con AAPH AAPH+Vit C
AAPH Groups

b B e
1500
B B B
Fluorescence Units/cell

1500

Fluorescence Units/cell
1000 A
1000 A

500
500

0 0
Con1 0mM 50mM Con ADVN ADVN+Vit C
ADVN Groups

c 2000
BC f
B
Fluorescence Units/cell

1600 1000
Fluorescence Units/cell

A B
800
1200 AC
AC 600
800 A
400

400
200

0 0
Con 0.5mM 2mM 8mM Con H2O2 H2O2+Vit C
H2O2 Groups

Fig. 1. Lipofuscin in erythrocytes of rats subjected to in vitro oxidative stress (a–c) and in cells treated with vitamin C (d–f). Values are expressed as
fluorescence units/ cell, n = 5. Changes as analyzed by one-way ANOVA followed by Tukeys post hoc test and represented in upper case at p < 0.05. Those
not sharing the same letters are significantly different.

noticeable in cells exposed to 10 and 50 mM, respectively of (Table 5). A similar trend was noticed when cells were incu-
ADVN. In contrast to AAPH, ADVN increased carbonyl bated with hydrogen peroxide. Increases were of equal
content by 72% for a 5-fold increase in its concentration. extent (40%) over the controls was evident in 2 mM as well
In the isolated membrane, carbonyl content increased by as 8 mM H2O2-exposed cells. A 16-fold increase in H2O2
337% (10 mM) and 436% (50 mM) in response to AAPH, concentration resulted in 11% increase in PrC (Table 3b).
and by 412% (10 mM) and 559% (50 mM) to ADVN. Increases in membrane PrC of 82% and 140% over the con-
Increases by 82% and 140% were seen in 2 mM and trols were seen in cells incubated with 2 mM and 8 mM
8 mM H2O2, respectively, (Table 3a). Treatment of cells H2O2, respectively, (Table 3b). Hence a 16-fold increase
with vitamin E and C reduced PrC levels by 12.2% and in H2O2, PrC increased 73%. In contrast to the azo
23%, respectively, over AAPH- and by 34% and 37%, compounds, H2O2 effect in increasing PrC levels were not
respectively, over ADVN-incubated cells. A 5-fold increase suppressed by antioxidants (Table 5). To determine if pro-
in AAPH and ADVN resulted in respective increases of tein sulphydryl oxidation was affected, another set of
22% and 28% in PrC. Antioxidants reduced PrC was by experiments were conducted to measure the sulphydryl
34% (vitamin E) and 37% (vitamin C), and by 43% (vitamin level in the membranes. P-SH levels showed a reduction
E and vitamin C) over AAPH and ADVN treated cells by 27% and 28% over the controls in 50 mM of AAPH
 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364 1361

Table 5 and ADVN-exposed cells, respectively. With a 5-fold

Effect of antioxidants on (a) azo-compound and (b) H2O2 – induced increase in AAPH and ADVN concentrations, P-SH
protein oxidation
reduced by 19% and 15% in the membrane (Fig. 2a and
AAPH AAPH + Vit C AAPH + Vit E b). P-SH reduced by 28% over the controls in cells exposed
(a) to 8 mM H2O2 and by 23% for a 16-fold increase in H2O2
PrC (L) 5.8 ± 0.6A 23.0 ± 0.1B 17.8 ± 0.8C 20.2 ± 0.2D concentration (Fig. 2c). Vitamin C and E did not have any
PrC (M) 5.6 ± 0.2A 30.2 ± 0.6B 19.1 ± 0.4C 19.8 ± 0.4C
signifying effect on stressed membrane sulphydryl content.
ADVN ADVN + Vit C ADVN + Vit E Since Band 3 is a major integral protein acting as a sensor
A B C
PrC (L) 5.8 ± 0.6 25 ± 0.2 20.2 ± 0.6 22.4 ± 0.5D for oxygen allocation within the erythrocyte, we separated
PrC (M) 5.6 ± 0.2A 37.1 ± 1.2B 20.6 ± 0.2C 21.5 ± 0.6C the protein and measured its level in the oxidatively
H2O2 H2O2 + Vit C H2O2 + Vit E stressed membrane. The intensity of band 3 decreased by
(b) 20% and 28% over the controls in the membrane of cells
PrC (L) 5.8 ± 0.6A 8.1 ± 0.1BD 7.1 ± 0.1AD 7.1 ± 0.2AD exposed to 10 mM and 50 mM, respectively, of AAPH
PrC (M) 4.3 ± 0.3A 7.9 ± 0.1B 7.1 ± 0.2B 7.2 ± 0.2B (Fig. 3a). At 10 mM and 50 mM, ADVN, reduction in
PrC, protein carbonyl; L, lysate; M, membrane. Values were expressed as band 3 intensity by 26% and 42% were evident (Fig. 3b).
mean ± SE of five animals/group. Cells were incubated individually with Enhanced protein oxidation was seen by 12% and 15%
AAPH/ADVN [50 mM] /H2O2 [2 mM] and vit C (60 mM); vit E (2 mM).
Changes between the groups are analyzed by one-way ANOVA followed
by Tukey–Kramer post hoc test. Values with different superscripts between
groups in each column are significantly different at p < 0.05 are repre- a 80

sented in upper case (ABC).

60
A

% Intensity
40 B
C
20

a 60 A AC 0
BC Con 10mM 50mM
AAPH
uM/mg protein

40
b 80
A
20 B
60 C
% Intensity

0
Con 10mM 50mM 40
AAPH
20
b 60
AC
uM/ mg protein

A BC 0
40
Con 10mM 50mM

20 ADVN

0 c 80

Con 10mM 50mM A

ADVN 60 B
C
c C
% Intensity

60 A A
BC
uM /mg protein

AC 40
40

20
20

0 0
Con 0.5mM 2mM 8mM Con 0.5mM 2mM 8mM
H2O2 H2O2

Fig. 2. Membrane sulfhydryl groups in erythrocytes of rats subjected to
in vitro oxidative stress (a–c). Values are expressed as mean ± SE of five
animals/group. Significance between groups is analyzed by Tukeys post
hoc test and represented in upper case at p < 0.05. Those not sharing the
same letters are significantly different.
Fig. 3. Band 3 in erythrocyte membrane of rats subjected to in vitro
oxidative stress (a–c). Values are expressed as mean ± SE of five animals/
group. Significance between groups is analyzed by Tukeys post hoc test
and represented in upper case at p < 0.05. Those not sharing the same
letters are significantly different.
1362 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364
for a 5-fold increase in concentration of AAPH and
ADVN, respectively. Oxidation of band 3 was also evident
in erythrocytes that were exposed to 0.5 (25%), 2 mM
(43%) and 8 mM (45%) H2O2 and increases were by 26%
for a 16-fold increase in H2O2 (Fig. 3c). A notable observa-
tion was that antioxidants did not have any significant
change in reversing the reduction in the intensity of band
3 and these were concomitant with the changes seen for
membrane sulphydryl concentration.
8. Discussion
The mechanisms by which the erythrocyte defends itself
against oxidative damage are very efficient and are located
in the cytosol and membrane domains. An interpretation
of our results on the mechanisms of oxidative stress
through in vitro models has to consider three factors,
namely, (1) the impact of free radical initiators comprising
water-soluble and lipid-soluble AAPH, H2O2, and ADVN
on hemolysis and osmotic fragility of erythrocytes, (2)
the influence of these compounds on lipid peroxidation
and protein oxidation in lysate and cell membrane
and (3) the effect of single antioxidants, vitamin E and
vitamin C in suppressing the mechanisms induced by free
radical initiators in the aqueous and lipid regions of
erythrocytes.
The observed increase in hemolysis for a 5-fold increase
in AAPH and ADVN concentration (10–50 mM) was
higher compared to a modest increase (27%) in response
to a 16-fold increase in H2O2 concentration (0.5–8 mM).
AAPH can generate free radicals at a constant rate in an
aquatic environment by unimolecular thermal decomposi-
tion without the addition of potentially interfering cofac-
tors and transition metals (Zou et al., 2001). In the
present investigation, erythrocytes showed increased fragil-
ity in response to ADVN (120% in 0.3% and 0.4% BSS) in
as low a concentration as 10 mM compared to a similar
concentration of AAPH (80% in 0.3% BSS). The extent
of increases was however low in spite of a 16-fold increase
in H2O2. H2O2 is not a free radical but is nonetheless highly
important because of its ability to penetrate biological
membranes (Nordberg and Arn’er, 2001). The observed
increased fragility due to azo compounds especially ADVN
may be explained in relation to increased lipid peroxidation
in the membranes. These findings are consistent with previ-
ous reports indicating a correlation with increased mem-
branous lipid peroxidation and decreased erythrocyte
deformability by AAPH (Begum and Terao, 2002). Earlier
studies have also suggested that osmotic fragility, a sensi-
tivity marker of change in osmotic pressure characteristic
of erythrocytes, is related to pathological situations, and
that these changes in cell integrity may be applied to the
diagnosis of hemolytic diseases (Kolanjiappana et al.,
2002).
Our results on increased MDA in membrane in response
to ADVN and AAPH suggest that in the face of a pro-
found external oxidative stress to the erythrocyte, the
plasma membrane is often the initial site of damage, and
that the resulting peroxidation of membrane lipids causes
hemolysis and cross-links between protein and lipid mole-
cules to different extents (May, 1998). Further, the
increased fragility leading to hemolysis is suggestive of
cytosolic enzymes being inefficient in reacting with ROS
generated in the membrane (Demehin et al., 2001).
The observed protective function of antioxidants in
reducing MDA is concomitant with the findings reported
by May (1998) on vitamin E and vitamin C in reducing
LPO. Vitamin C not only directly consumes oxidant free
radicals in plasma, but also protects and recycles vitamin
E in lipoproteins and erythrocytes. Vitamin C does not
directly affect the membrane LPO, but it may perform this
function indirectly by reducing the tocopheroxyl free radi-
cals in the lipid bilayer and the mechanism presumably
involves a reduction of the a-tocopheroxyl free radicals at
the aqueous-lipid interface of the membrane bilayer. In
doing this, it protects the cell membrane and decreases
hemolysis.
The H2O2 generated during the autoxidation of oxyhe-
moglobin has been shown to initiate a cascade of reactions
resulting in the formation of fluorescent degradation prod-
ucts (Nagababu and Rifkind, 2000). Further, the mecha-
nism increasing the toxic potency of H2O2 is a modified
Haber–Weiss reaction that involves the direct reduction

of H2O2 by O 
2 , with the formation of O2 , and OH
(Djordjevic, 2004). The toxicity of H2O2 is largely due to
its ready conversion to the reactive hydroxyl radical
(OH), either by exposure to ultraviolet light or by interac-
tion with a range of transition metal ions, of which the
most important in vivo is probably iron. Our finding of a
higher lipofuscin content in H2O2 – than in ADVN – trea-
ted cells perhaps supports the view that H2O2 can contrib-
ute to Fenton chemistry not only by being one of the
substrates but also by providing another, e.g. by liberating
iron from heme proteins (Halliwell et al., 2000).
The head group of vitamin E, facing towards the surface
of the membrane, is responsible for scavenging free radi-
cals, whereas the function of the side chain is to retain
the vitamin E molecule in the membrane (Van Acker
et al., 1993). Thus, free radicals generated by compounds
such as ADVN in the lipid moiety are in contact with the
phytyl chain and are farther away from the vitamin E head
group, which is essential in the scavenging process. Con-
versely, the free radical scavenging head group at the sur-
face would be optimal for protecting against free radicals
generated in the aqueous phase. Our findings on vitamin
E scavenging properties being significantly better when
the free radicals are generated in the aqueous phase by
AAPH as opposed to those generated in the lipid phase
by ADVN are somewhat similar to those reported for
AMVN (Massaeli et al., 1999). They also suggest that vita-
min E and vitamin C can protect cells from lipid peroxida-
tion. The benefits derived from vitamin E in human
erythrocytes when challenged with phenylhydrazine hydro-
chloride have been reported by Claro et al. (2006).
 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364
The present study demonstrates an increased oxidation
of proteins in membranes rather than in lysates with the
extent of oxidation being higher in cells exposed to azo
compounds than H2O2. AAPH predominantly attacks the
erythrocyte membranes from the cell exterior and the per-
oxyl radicals generated remain in the interior of the mem-
brane bilayer. However, at a certain stage during
oxidation, these radicals permeate the cells (Zou et al.,
2001).
Our results on a 15–19% decrease in membranous P-SH
for a 5-fold increase in AAPH and ADVN (10–50 mM) as
compared to a 23% decrease in response to a 16-fold
increase in H2O2 suggest increased oxidation of the sulfhy-
dryl (SH) group to disulphides in the membrane. They also
support earlier findings on the role of azo compounds on
an increased permeability of hydrophilic nonelectrolytes
and ions, a trans-bilayer orientation of phospholipids and
finally a decreased cell deformability (Xia et al., 1999)
and the reactivity of H2O2 with heme proteins and pro-
tein-SH groups (Halliwell and Gutteridge, 1999). Although
vitamin E and vitamin C are potential scavengers of per-
oxyl radicals, the oxidation of surface thiols of extrinsic
proteins is not inhibited (Zou et al., 2001), which is also evi-
dent from our results on an insignificant increase of the
sulfhydryl groups in response to the antioxidants.
Oxidative modification of proteins, considering their
multiple functions, unlike lipid peroxidation, can be selec-
tive and specific (Dubinina et al., 2002). Our results on
reduced SH level in the membrane are suggestive of a pro-
motion of oxidation by extra cellular oxidants and are con-
comitant with a decreased intensity of band 3 protein in the
membrane. They are also supportive of the reports on oxi-
dation of SH groups leading to a change in the protein con-
formation that result in loss of binding sites and affinity for
oxygen (Xia et al., 1999).
Further, our data on reduced band 3 protein by H2O2 as
well as by the azo compounds are in accordance with those
reported by Sato et al. (1995) on LPO and conformational
change of band 3 being responsible for increased hemolysis
induced by peroxyl radicals. AAPH-induced oxidation of
band 3 is reported to induce pore formation and hemolysis
(Simao et al., 2006).
A noteworthy finding in the present study is that incuba-
tion of rat erythrocytes at a 5-fold AAPH or ADVN
resulted in higher protein and membrane oxidation com-
pared to a 16-fold increase in H2O2. Our findings suggest
that neither protein sulphydryl oxidation nor band 3 by
peroxyl radical-mediated mechanisms could be inhibited
by vitamin E or vitamin C unlike protein carbonyl oxida-
tion in the membrane.
In conclusion, the results of our study revealed a higher
oxidant capacity of azo compounds than hydrogen perox-
ide, thereby suggesting that AAPH and ADVN are efficient
models to study the mechanisms of oxidative injury result-
ing in situations such as hypoxia, blood storage, in a trans-
fusion setting and in anemia where oxidative stress induces
premature clearance of erythrocytes from the circulation.
1363
Our study also emphasizes the possibility of utilizing the
in vitro models for evaluating the benefits of a combination
of vitamin E and vitamin C while evaluating the mecha-
nisms of oxidative injury in the two phases. However, it
remains to be studied whether membrane sulphydryl and
band 3 oxidation can be altered by antioxidants in combi-
nation, and whether such a combination would also be
effective in reducing hemolysis and osmotic fragility under
various physiological conditions.
Acknowledgements
The present study was supported by the grants received
by the Department of Science and Technology (SP/SO/AS-
58/2004), New Delhi to Ms. S. Asha Devi.
References
Asha, D.S., Subramanyam, M.V.V., Vani, R., Jeevaratnam, K., 2005.
Adaptations of the antioxidant system in erythrocytes of trained adult
rats: Impact of intermittent hypobaric-hypoxia at two altitudes.
Comparative Biochemistry and Physiology. Part C 140, 59–67.
Asha, D.S., Vani, R., Subramanyam, M.V.V., Shankar, S.R., Jeevarat-
nam, K., 2007. Intermittent hypobaric hypoxia-induced oxidative
stress in rat erythrocytes: protective effects of vitamin E, vitamin C and
carnitine. Cell Biochemistry and Function 24, 1–11.
Begum, A.N., Terao, J., 2002. Protective effect of alpha-tocotrienol
against free radical-induced impairment of erythrocyte deformability.
Bioscience Biotechnology Biochemistry 66, 398–403.
Claro, L.M., Leonart, M.S.S., Comar, S.R., Nascimento, A.J., 2006.
Effect of vitamins C and E on oxidative processes in human
erythrocytes. Cell Biochemistry and Function 24, 531–535.
Demehin, A.A., Abugo, O.O., Rifkind, J.M., 2001. The reduction of nitro
blue tetrazolium by red blood cells: a measure of red blood cell
membrane antioxidant capacity and hemoglobin-membrane binding
sites. Free Radical Research 34, 605–620.
Djordjevic, V.B., 2004. Free radicals in cell biology. International Review
of Cytology 237, 57–89.
Dodge, J.T., Mitchell, C., Hanahan, D.J., 1963. The preparation and
chemical characteristics of hemoglobin-free ghosts of human erythro-
cytes. Archives Biochemistry and Biophysics 100, 119–130.
Dubinina, E.E., Gavrovskaya, S.V., Kuzmich, E.V., Leonova, N.V.,
Morozova, M.G., Kovrugina, S.V., Smirnova, T.A., 2002. Oxidative
modification of proteins: oxidation of tryptophan and production of
dityrosine in purified proteins using Fenton’s system. Biochemistry
(Moscow) 67, 343–350.
Fujino, T., Watanabe, K., Beppu, M., Kikugawa, K., Yasuda, H., 2000.
Identification of oxidized protein hydrolase of human erythrocytes as
acylpeptide hydrolase. Biochimica Biophysica Acta 1478, 102–112.
Habeeb, A.F.S.A., 1972. Reaction of protein sulfhydryl groups with
Ellman’s reagent. Methods in Enzymology 34, 457–464.
Halliwell, B., Clement, M.V., Long, L.H., 2000. Hydrogen peroxide in the
human body. FEBS Letters 486, 10–13.
Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and
Medicine, 3rd ed. Clarendon Press, Oxford.
Kolanjiappana, K., Manoharana, S., Kayalvizhib, M., 2002. Measurement
of erythrocyte lipids, lipid peroxidation, antioxidants and osmotic
fragility in cervical cancer patients. Clinical Chimica Acta 326, 143–149.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227, 680–685.
Liu, Z., Han, K., Lin, Y., Luo, X., 2002. Antioxidative or prooxidative
effect of 4-hydroxyquinoline derivatives on free-radical-initiated hemo-
lysis of erythrocytes is due to its distributive status. Biochimica
Biophysica Acta 1570, 97–103.
1364 C.S. Shiva Shankar Reddy et al. / Toxicology in Vitro 21 (2007) 1355–1364
Lowry, O.H., Rosenberg, N.J., Farr, A.L., Randall, R.J., 1951. Protein
measurements with Folin-phenol reagent. Journal of Biological
Chemistry 193, 364–375.
Massaeli, H., Sobrattee, S., Pierce, G.N., 1999. The importance of lipid
solubility in antioxidants and free radical generation generating
systems for determining lipoprotein peroxidation. Free Radical Biol-
ogy Medicine 26, 1524–1530.
May, J.M., 1998. Ascorbate function and metabolism in the human
erythrocyte. Frontiers in Bioscience 3, 1–10.
Nagababu, E., Chrest, F.J., Rifkind, J.M., 2003. Hydrogen-peroxide-
induced heme degradation in red blood cells: the protective roles of
catalase and glutathione peroxidase. Biochimica Biophysica Acta 1620,
211–217.
Nagababu, E., Rifkind, J.M., 2000. Reaction of hydrogen peroxide with
ferrylhemoglobin: superoxide production and heme degradation.
Biochemistry 39, 12503–12511.
Nordberg, J., Arn’er, E.S.J., 2001. Reactive oxygen species, antioxidants,
and the mammalian thioredoxin system. Free Radical Biology
Medicine 31, 1287–1312.
O’Dell, B.L., Browning, J.D., Reeves, P.G., 1987. Zinc deficiency increases
the osmotic fragility of rat erythrocytes. Journal of Nutrition 117,
1883–1889.
Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxidation in
animal tissues by thiobarbituric acid reaction. Analytical Biochemistry
95, 351–358.
Okamoto, K., Maruyama, T., kaji, Y., Harada, M., Mawatari, S., Fujino,
T., Uyesaka, N., 2004. Verapamil prevents impairment in filterability
of human erythrocytes exposed to oxidative stress. Japanese Journal of
Physiology 54, 39–46.
Sato, Y., Kamo, S., Takanishi, T., Suzuki, Y., 1995. Mechanism of free
radicals-induced haemolysis of human erythrocytes: haemolysis by
water-soluble radical initiator. Biochemistry 34, 8940–8949.
Senturk, U.K., Gunduz, F., Kuru, O., Aktekin, M.R., Kipmen, D.,
Yalcin, O., Kucukatay, M.B., Yesilkaya, A., Baskurt, O.K., 2001.
Exercise-induced oxidative stress affects erythrocyte in sedentary rats
but not exercise-trained rats. Journal of Applied Physiology 91, 1999–
2004.
Simao, colado An., Suzukawa, A.A., Casado, M.F., Oliveira, R.D.,
Guarneir, F.A., Cecchini, R., 2006. Genistein abrogates pre-hemolytic
and oxidative stress damage induced by 2,2 0 -azobis (amidinopropane).
Life Sciences 78, 1202–1210.
Uchida, K., Stadtman, E.R., 1993. Covalent attachment of 4-hydroxy-
nonenal to glyceraldehyde-3-phosphate dehydrogenase. Journal of
Biological Chemistry 268, 6388–6393.
Van Acker, S.A., Koymans, L.M., Bast, A., 1993. Molecular pharmacol-
ogy of vitamin E: structural aspects of antioxidant activity. Free
Radical Biology Medicine 15, 311–328.
Xia, J., Browning, J.D., O’Dell, B., 1999. Decreased plasma thiol
concentration is associated with increased osmotic fragility of eryth-
rocytes in zinc-deficient rats. Journal of Nutrition 129, 814–819.
Zou, C.G., Agar, N.S., Jones, G.L., 2001. Oxidative insult to human red
blood cells induced by free radical initiator AAPH and its inhibition by
a commercial antioxidant mixture. Life Sciences 69, 75–86.