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Changes in Zinc Levels

and Superoxide Dismutase Activities
in the Skin of Acute, Ultraviolet-B-
Irradiated Mice After Treatment
With Ginkgo Biloba Extract
AYSEL ARICIOGLU,*,1 MERAL BOZKURT,2
BARBAROS BALABANLI,1 MEHTAP KıLıNÇ,3
N. KEMAL NAZAROGLU,1 AND NURTEN TÜRKÖZKAN1
Departments of 1Biochemistry, 2Dermatology, and
3
Pharmacology, Medical Faculty, Gazi University,
06510 Besevler, Ankara,Turkey
Received June 6, 2000; Revised October 30, 2000;
Accepted November 11, 2000

ABSTRACT
Acute ultraviolet-B (UV-B) irradiation is known to act as an ini-
tiator in the formation of reactive oxygen species. These oxygen prod-
ucts are highly reactive and they are able to cause irreversible damage
to cellular components. Oxygen free radicals are normally neutralized
by very efficient systems in the body. These include antioxidant
enzymes like superoxide dismutase (SOD). In a healthy subject, there
is a balance between free radicals and the levels of antioxidants. In
some pathological conditions such as oxidative stress, the level of
antioxidants is significantly reduced. The skin contains relatively high
levels of zinc (Zn), an essential element known to be a cofactor in
some metabolic pathways. Zinc has also been reported to have anti-
oxidant properties.
In the present study, we investigated the effect of ginkgo biloba
extract (Gbe), a potent free-radical scavenger, on UV-B-irradiated skin
by measuring SOD activity and Zn levels in the skin, before and after
treatment. The SOD activity was decreased after UV-B exposure, in
comparison with the control group (p < 0.05). After Gbe treatment,

*Author to whom all correspondence and reprint requests should be addressed.

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176 Arıcıoglu et al.

the SOD activity increased (p < 0.05) as compared with the untreated
UV-B irradiated group. The Zn levels changed in the same pattern as
the SOD activity values.
Index Entries: Superoxide dismutase; zinc; UV-B; ginkgo biloba
extract.

INTRODUCTION
Exposure of the skin to ultraviolet-B (UV-B) radiation induces the
photochemical generation of reactive oxygen species (ROS). These in-
clude the superoxide radical (O•2−), the hydroxyl radical (OH•), hydrogen
peroxide (H2O2) and organic peroxides (1–3).
The enzyme superoxide dismutase (SOD) catalyzes the dispropor-
tionation of the reactive O•2− species into dioxygen and oxygen peroxide,
thereby protecting cells against ROS by scavenging of the superoxide
radicals produced by ionizing radiation or by other mechanisms (4,5).
The epidermal concentrations of zinc, which is a constituent of a vari-
ety of enzymes, including SOD, are changed in patients with dermato-
logical diseases (6). Zinc also has an indirect antioxidant role in cells (1,7).
The ginkgo biloba extract (Gbe), among other compounds, contains
flavonoids, which have been reported to exhibit potent free-radical scav-
enging activities (8,9). The purpose of this study was to investigate the
effects of Gbe on total SOD activity and Zn levels in the skin of mice after
acute UV-B irradiation.

MATERIALS AND METHODS

Inbred BALB/C, 5-mo-old albino female mice were used in our
experiments. The mice were divided into four groups, each group was
composed of six mice. These were classified as follows:
Group I: Control group
Group II: Exposed to acute UV-B irradiation
Group III: Received 100 mg Gbe/kg/d p.o. for 5 d prior to
irradiation
Group IV: Received a single oral dose of 100 mg Gbe/kg
immediately after irradiation
The backs of the mice were shaved before being exposed to a single
dose of 0.24-J/cm2 UV-B irradiation for 8 min. The animals were sacri-
ficed by cervical dislocation 18 h after irradiation.
The dorsal skin of each mice was carefully dissected and immedi-
ately put into liquid nitrogen to be stored at −70ºC until needed. The tis-
sues were homogenized and then sonicated to ensure homogeneity. The
total SOD activity and the Zn levels were determined in the collected
supernatant of tissue homogenates.

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Zinc and SOD in Skin of Irradiated Mice 177

Table 1
Superoxide Dismutase Activity and Zinc Levels in Four Study Groups*

*Significant differences in SOD activities between groups I and II, I and III,
I and IV, II and III, and II and IV were set at p < 0.05. No significant differences
were found in SOD activities in groups III and IV. There were no significant dif-
ferences for the zinc levels in any of the groups.
**Values are the mean ± SEM.

The SOD activities were measured by the nitroblue tetrazolium inhi-

bition assay (10). The results were expressed as unit per milligram of pro-
tein. The protein concentration was measured by the method of Lowry
et al. (11). The zinc concentrations were determined by atomic absorption
spectrometry and the results were expressed as microgram per milligram
of protein.
All results are expressed as mean ± SEM of each group; the statisti-
cal significance was determined by analysis of variance (ANOVA).

RESULTS
The results of this study are summarized in Table 1. The SOD activ-
ities in groups II, III, and IV were decreased when compared with those
of group I. When compared with group II, the SOD activities of groups
III and IV were found to be increased.
The tissue zinc levels did not show significant differences among
groups but followed parallel increases or decreases with SOD activities.

DISCUSSION
Oxygen radical species produced by UV-B exposure have been
identified as the cause of cellular oxidative damage (12). There are

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178 Arıcıoglu et al.

pathological conditions in which oxidative stress causes the levels of

antioxidants to fall below their normal values (13). A single dose of UV
irradiation leads to an impairment of the antioxidant defense system,
although there are reports indicating that long-term in vivo UV exposure
and low UV-B doses result in an induction of SOD activity (14–16).
Another study suggests that UV light is an important regulatory factor
in the skin antioxidant defense mechanism (17).
In this study, the decrease in SOD activities 18 h after acute UV-B
irradiation in the untreated group might be attributed to its degradation
by UV-B-induced ROS. Some antioxidants can be destroyed directly by
UV light but SOD is quenched by an indirect mechanism that involves
free radicals and its oxidation product, H2O2 (18,19). In addition, in some
studies the SOD-induction capacity in response to oxygen stress was
found to be lower in rodents (20,21). Therefore, for that time-point (18th
h), the decrease in SOD activity can be the result of either a low enzyme-
induction capacity or an enzyme degradation process.
Decreased Zn concentrations have been found in the epidermis of
subjects with dermal pathological conditions (6). Zinc also plays a major
role in the stabilization of cell membranes by protecting —SH groups
and inhibiting the formation of free radicals by competition with other
transition metal ions (3).
In all groups in this study, the tissue Zn levels increased or de-
creased in parallel with SOD activities. Another study reported that x-ray
irradiation could induce redistribution of Zn in skin (6). Berger et al.
reported that Zn skin levels could be depressed in burns (22). The
decrease in Zn levels in group II with respect to group I might be attrib-
uted to the redistribution of Zn and, therefore, to a loss in skin Zn con-
tent after acute UV-B irradiation.
In groups III and IV, those treated with Gbe, we found increases in
both SOD activities and Zn levels when compared with those of the irra-
diated, untreated group II. Because it is known that ROS induced by the
UV-B irradiation triggers lipid peroxidation, which also induces forma-
tion of lipid peroxide radicals (14), treatment with Gbe might work by
scavenging ROS, thus decreasing tissue oxidative damage. Therefore,
although the increase in SOD activities was probably the result of
decreased enzyme degradation, the increase in Zn levels may be the
result of a preventive effect of Gbe from oxidative damage.
From our results, it may be concluded that Gbe treatment leads to close
to normal SOD activity and zinc levels in the skin of UV-B-irradiated mice.

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