Rom. J. Biophys. 2002
12(3-4):103-115
ANTIOXIDANT ACTIVITY OF
ASCORBIC ACID AGAINST PEROXIDATION OF PHOSPHATIDYLCHOLINE LIPOSOMES EXPOSED TO
GAMMA RADIATION: A SYNERGISTIC INTERACTION?
M.H. GABER*, N.A. ABD EL HALIM, W.A. KHALIL
*Department of Biophysics, Faculty of Science,
Central Lab for NMR, Faculty of Science,
Abstract. Ionizing radiation induced
damage of cellular membrane is known to alter many structural and physiological
processes leading to the loss of the normal cellular function including cell
death. The present study was designed to determine the changes in bilayer
permeability in egg lecithin multilamellar vesicles after exposure to
gamma-radiation at doses of 500 Gy and 1 kGy. Liposomal changes in permeability
were monitored by measuring the leakage of pre-encapsulated
6-carboxyfluorescein (CF). The changes in permeability in the bilayer were
found to be dependent on radiation dose. The presence of hydrophilic
antioxidant ascorbic acid (0.1 mM) in the aqueous medium drastically increased
the leakage of carboxyfluorescein from liposomes. IR and NMR studies which have
been employed to reveal structural alterations in irradiated vesicles showed an
increased damage upon exposure to radiation in the presence of ascorbic acid.
The proton nuclear magnetic resonance (1H NMR) spectrum of the
irradiated lipid vesicles (in the presence of ascorbic acid) showed a
preponderance of signals attributable to new compounds formed as a consequence
of the degradation process. The obtained results contribute to our
understanding of the mechanism of radiation oxidative damage and its
modification by radical scavenging and/or organizational modulation, which
emphasize the importance of structure and composition of antioxidant in
developing new strategies to reduce the damaging effects of ionizing radiation.
Key words: Gamma-radiation, liposome,
carboxyfluorescein, ascorbic acid, nuclear magnetic resonance, infrared
spectroscopy, fluorescence, electron microscopy.
INTRODUCTION
The
concept of cell membrane phospholipids has turned from their passive,
structural role in the buildup of structural components [29] to the more
active, functional regulation of cell functions [10, 25, 32}.
They are now considered a vital element in initiating a variety of cell
functions. One of the most important roles is their function as intracellular
second messengers. The second messengers are essential in the control of
cellular functions, like stimulation of cell proliferation and differentiation.
There is now increasing evidence to suggest that choline phospholipids may
function as second messengers and may be involved in activation of mitogenic
signal transduction [1, 5, 9]. For these reasons, the
experimental investigation of the radiation effects at the membrane level is of
high scientific interest.
Oxidative
damage of membrane is mediated by the free radicals generated in the cytosol
and in the surrounding of the cell, which are believed to be involved in
radical chain reactions [22]. Polyunsaturated fatty acyl chains in biological
membrane are the critical targets, which display susceptibility to attack by
the radiation induced free radicals. Liposomes offer a suitable model membrane
system to investigate the molecular mechanism of membrane damage induced by
radiation since the composition of liposomes can be easily controlled and
varied [31]. Previous results have demonstrated that the presence of
cholesterol in the composition of egg lecithin liposomal membrane produced a
modulating effect on structural order of the membrane bilayer, which reflected
in significantly protecting liposomes against radiation damage.
The
antioxidant properties of ascorbic acid and of its lipophilic derivatives have
been the object of several papers [6, 13]. However, there is suspicion that
ascorbic acid can both act as a strong, efficient, and cheap antioxidant agent
on one level, and at the same time behaves as a
radical promoter on a different level, and produces dangerous species in living
systems [16].
This
report describes results on radiation effects on liposomes prepared from egg
phosphatidyicholine (Egg Pc) and concerned the protective effect of ascorbic
acid as a valuable antioxidant material. We have employed a fluorescence probe,
carboxyfluorescein (CF), to detect the changes in permeability of liposomal
membrane [23]. It is shown that irradiation induced membrane damage exploited
by the increased release of the fluorescent material with radiation dose.
Results have further shown that ascorbic acid synergizes the effect of
radiation by enhancing the release of carboxyfluorescein from liposomes.
Nuclear magnetic resonance and infrared study confirmed the synergistic effect
of ascorbic acid in protecting the damage to liposomal membrane against gamma
radiation.
MATERIALS AND METHODS
MATERIALS
Egg
phosphatidylcholine in carboxyfluorescein, and ascorbic acid were procured from
Sigma. Sephadex G‑50 coarse was a product of
Pharmacia Biotech. Aqueous solutions were prepared in distilled water.
Fluorescence and infrared spectroscopy (IR) measurements were made using
phosphate buffer saline (PBS) filtered through 0.22 micrometer membrane filter.
Organic solvents (ethanol, chloroform, ethyl acetate, diethyl ether) were
obtained from Merck (
METHODS
Preparation of multilamellar
liposomes encapsulating CF
Liposomes
were prepared by the film hydration method [14]. Phospholipids were dissolved
in chloroform in a rounded bottom flask. The organic solvent was evaporated
under vacuum leaving a uniform thin film on the inner walls of the flask. The
film was hydrated with I ml of 10 mM PBS (pH 7.4) to give a lipid concentration
of 10 mmol/ml. Multilamellar liposomes were formed by constant vortexing for 4
min in a vortex mixer and sonication in a bath sonifier for 10 min. During
hydration, the temperature of egg Pc liposome suspension was maintained at 45
°C.
Ascorbic
acid was dissolved in PBS buffer at a concentration of 0.1 mM and mixed with
the liposome solution.
To
encapsulate CF in egg Pc vesicles, the lipid film was hydrated with 100 mM CF
solution prepared in PBS followed by usual procedure of sonication. The
multilamellar vesicles obtained after sonication were filtered through Sephadex
G-50-gel minicolumn to separate the non-encapsulated probe material [15].
Irradiation of liposomes
The
multilamellar liposomes prepared from egg Pc were irradiated at room
temperature by 137Cs g-rays (500 Gy, 1 kGy) at the National
Institute of Standards (
Scanning electron microscopy
Scanning
electron microscopy was performed with a JEOL Model JEM 100 S/Japan, capable of
magnification up to x 200,000, a resolution power of 5 nm, and accelerating
voltage from 40 to 100 kV. Vesicle size distribution was determined by
measuring the diameter of fractured vesicles as previously reported [11]. The
vesicle average diameter was 200 nm.
Determination of vesicle
permeability
Membrane
permeability in irradiated egg Pc vesicles was monitored by the leakage of CF
from liposomes after exposure to a dose of 500 Gy and 1 kGy, followed by
incubation at room temperature up to 3 h. Self-quenched concentration of CF
(> 50 mM) in liposomes developed an intense fluorescence when it was diluted
after leakage in outer aqueous environment. The fluorescence intensity of CF
was measured at room temperature (lex = 490 nm, and lem = 520 nm) by
spectrofluorimeter type Schimadzu. Permeability of liposornes was expressed in
terms of relative fluorescence intensity, which was the ratio between intensity
of a particular liposome sample and the total fluorescence obtained by
disruption of liposomes, by Triton X-100 (0.01%) and multiplied by 100.
Iinfrared measurements
A
film of small amount of liposome sample (~ 2 ml) was deposited between two
disks of potassium bromide, avoiding the presence of air. For each sample the
spectra were recorded 3 times with 16 scans from 4000 to 1000 cm1
on a Jasco model v-570 infrared spectrometer.
NMR measurements
1H NMR measurements were
carried out on an Oxford XL-300 at 300 MHz. Between 16 and 128 scans were used,
The 1H chemical shifts are reported in ppm relative to
tetramethylsilane (d 0.0) as internal reference. All measurements were performed at room
temperature. The solvent used was CDCl3.
RESULTS
CHARACTERIZATION OF
LIPOSOMES USED
Liposomes
formed by egg Pc were prepared by using the hydration method yielding 200 nm
liposomes. The vesicles were analyzed by electron microscopy. Figure 1 shows
the size distribution analysis of the resultant liposome sample.
CF FLUORESCENCE 1N
IRRADIATED VESICLES
Egg
phosphatidylcholine liposomes encapsulated with CF were g-irradiated at two radiation
doses (500 Gy & 1 kGy). The fluorescence intensity of liposomes measured
after 3 hours of incubation at room temperature showed that the degree of CF
leakage from the Pc liposomes was dependent on the applied radiation dose as is
clear from Figure 2. The percent release of CF from vesicles irradiated at 500
Gy dose was 30 times higher compared to control, which was found to increase at
a higher radiation dose (1 kGy). These findings are clearly illustrated in
Figure 3.
Addition
of ascorbic acid to the buffer medium at a concentration of 0.1 mM caused a
dramatic increase in the fluorescence intensity upon gamma-irradiation. The
increase in fluorescence intensity was beyond the measurable level, and
therefore the precise percent of release could not be calculated.
Fig. 1. Size distribution of egg Pc vesicles obtained from electron
microscopy.
Fig.
2 Schematic fluorescence emission spectra of 5(6)-carboxyfluorescein obtained
on three differently treated liposome samples in a 0.5 cm quarts cuvette.
Fig.
3. Effect of y radiation dose on the release of
5(6)-carboxyfluorescein from liposomes composed of egg Fe.
INFRARED MEASUREMENTS
The
infrared spectra of membrane lipids can be separated to great advantage into
spectral regions, which originate from molecular vibrations of the hydrophilic
head-group and those of the hydrophobic hydrocarbon tail. Figure 4a shows the
IR spectrum of control (non-irradiated) egg Pc liposome sample. The internal
vibrations of the lipid acyl chains are readily assigned on the basis of the
well- known dispersion curves of polymethylenes [8] and by comparison with
infrared spectra of fatty acid esters and other polymethylene-chain compounds
[4].
The
IR spectrum in Figure 4.a shows a clear peak at 3480 cm1 and
another peak at 1640 cm1. The first peak is due to the
carbon-hydrogen-stretching vibration. This peak is generally the strongest band
in the spectra of lipids and the frequencies of these bands are conformation
sensitive [3]. The second peak is characteristic for the head group modes [7]
particularly the C=O stretching band.
Irradiation
of the lipids at 500 Gy dose (Fig. 4b) caused a pronounced change in the
frequencies corresponding to acyl chain conformation (decrease in frequency
from 3480 to 3460 cm‑1) and in the frequencies corresponding
to head group modes (increase in the frequency from 1640 to 1650 cm1).
Addition
of ascorbic acid to the liposome sample during y irradiation at 500 Gy caused a
dramatic decrease in the frequency of the first band to 3400 cm1
and an increase in the frequency of the second band to 1660 cm1.
Fig. 4. Infrared spectra of aqueous dispersions of egg Pc exposed to
different doses of gamma radiation. a. For control non-irradiated liposome sample;
b. for samples exposed to 500 Gy in the presence and absence of 0.1 mM ascorbic
acid; c. for samples exposed to 1 kGy in the presence and absence of 0.1 mM
ascorbic acid.
Fig. 5. The 300 MHz 1H NMR (CD Cl3) spectrum of the
non-irradiated liposome sample composed of egg Pc.
Irradiation
of the lipid at l kGy dose (Fig. 4c) did not show any change in the frequency
band of the first peak. However, an increase in the second band frequency to
1650 cm1 was indicated.
Addition
of ascorbic acid to the liposome sample during g-irradiation caused an
increase of the frequency of the second band up to 1660 cm1 without
any effect on the first frequency band (3480 cm1).
NMR MEASUREMENTS
As
Pc was the major component of all the liposomes, signals from this lipid served
as a reference for comparing the intact liposomes with those irradiated with
gamma radiation. Figure 5 shows the 1H NMR spectrum of intact Pc
from a control non-irradiated sample. The singlet signal at 3.3 ppm corresponds
to (CH3)3N+, a multiplet at ≈ 3.7 ppm assigned to
CH2N+, and the signal at 4 ppm, attributable to the CHO
group. All the signals observed and indicated on the graph were assigned with
the help of references and NMR-tables [17, 18].
Irradiating
the lipids with gamma radiation at a dose of 500 Gy displayed the 1H
NMR spectrum in Figure 6a. The spectrum shows the disappearance of the peaks
from 1.5 3 ppm and the appearance of a new singlet peak at 4.658 ppm.
Fig. 6. The 300 MHz 1H NMR
(CD Cl3 ): a. the spectrum of liposome sample exposed to 500 Gy g radiation dose; b. the
spectrum of the liposome sample exposed to the same radiation dose in the
presence of 0.1 mM ascorbic acid.
Fig. 7. The 300 MHz 1H NMR (CD Cl3): a. the spectrum
of liposome sample exposed to 1 kGy g radiation dose; b. the spectrum of the
liposome sample exposed to the same radiation dose in the presence of 0.1 mM
ascorbic acid.
Irradiating
the lipids with gamma radiation at the same dose and in the presence of
ascorbic acid (0.1 mM) displayed the 1H NMR spectrum in Figure 6b.
The spectrum shows the disappearance of the peaks in the region from 3 4 ppm.
These are attributable to new compounds having been formed as a consequence of
the degradation process originated from gamma irradiation.
More
and more changes in the NMR spectrum were detected when the lipids were exposed
to a radiation dose of 1 kGy, as indicated in Figure 7a. The presence of
ascorbic acid (0.1 mM) during the irradiation procedure (Fig. 7b) shows a
strong sharp peak at 4.685 ppm that could be attributed to the oxidation of the
double bonds.
DISCUSSION
The
behavior of cell growth and its transmembrane properties post-irradiation is
one of the most interesting studies in biological sciences [28, 30]. However,
the mechanism of radiation induced effects in biological membrane and
subcellular components seem to be highly complicated.
Oxidative
damage of cellular membrane by ionizing radiation has been known to cause
impairment of the functional behavior of membrane and also of enzymes and
receptors [22]. Modification of damage at membrane level offers an important
strategy for the protection of normal cells against unavoidable
therapeutic/accidental radiation exposure.
Model
membranes, such as phospholipid liposome bilayers, are simple two- dimensional
systems with well-characterized physical properties at their different phases
[20]. Therefore, our study aimed to understand the structural behavior of
phospholipid molecules presented in liposome vesicles upon exposure to gamma
irradiation. The results also elucidated the protective role and antioxidant
activity of ascorbic acid when exposed to ionizing gamma radiation.
Alterations
in membrane fluidity in irradiated model [12, 19] as well as cellular membrane
[24] have been reported employing DPH fluorescence probe. Moderate doses of
gamma radiation are known to significantly increase membrane permeability of
HeLa cells [31]. The increase in leakage of CF fluorescence up to a dose of 1
kGy suggests a radiation induced increase in bilayer permeability. On the other
hand, addition of ascorbic acid during irradiation procedure increased the
amount of CF released from liposomes. These results suggest that for a defined
radiation dose, greater membrane damage occurs when using a hydrophilic
antioxidant material.
The
results obtained from IR and NMR demonstrated the presence of hydrolytic and
oxidative processes, which was enormously enhanced in the presence of ascorbic
acid.
Ascorbic
acid has been shown to be rapidly destroyed by gamma radiation in dilute
solutions [26]. The destruction product is called dehydroascorbic acid [21].
Both ascorbic and dehydroascorbic acids have biological activity. But these
biological activities differ [16]. Only ascorbic acid has the typical vitamin C
working while the breakdown product dehydroascorbic acid is highly unstable and
has no vitamin C working. If through circumstances high levels of
dehydroascorbic acid are formed, then it can even damage a number of biological
processes and affect health adversely [2, 27]. Our study suggests that ascorbic
acid should be protected from radiation in order to perform its protective role
adequately; perhaps this could be employed by encapsulating ascorbic acid in
the liposome vesicles.
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