Connective Tissue Research, 51:55–58, 2010

Copyright

c Informa UK Ltd.
ISSN: 0300-8207 print / 1607-8438 online
DOI: 10.3109/03008200902998709

Effect of Date Seed Oil on p53 Expression in Normal

Human Skin
Ines Dammak
Unité de Recherche, Pathologies Humaines et Stress Oxydatif, Institut Supérieur de Biotechnologie de
Sfax, Sfax, Tunisie
Sonia Boudaya
Connect Tissue Res Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 11/14/14

Service de Dermatologie, Centre Hospitalo-Universitaire, Sfax, Tunisie

Fatma Ben Abdallah
Unité de Recherche, Pathologies Humaines et Stress Oxydatif, Institut Supérieur de Biotechnologie de
Sfax, Sfax, Tunisie
Hamida Turki
Service de Dermatologie, Centre Hospitalo-Universitaire, Sfax, Tunisie
Hamadi Attia
Unité d’Analyses Alimentaires, Département de Biologie, Ecole Nationale d’Ingénieurs de Sfax,
Sfax, Tunisie
For personal use only.

A critical step to escape from the carcinogenic potential of UV
radiation is mediated by the protein p53. P53 activates growth
arrest, allowing for DNA repair, which removes damaged cells.
The concept of photoprotection involves blocking apoptosis and
the prevention of oxidative damage to cellular DNA. Date seed oil
(DSO) extract has been reported to be beneficial in the reduction
of chemically induced oxidative stress in normal human skin. In
this study, we investigated the DNA-protective qualities of DSO
as measured by p53 expression in human skin biopsies, one day
after exposure to ultraviolet B (UVB) radiation. P53 expression
was analyzed using immunohistochemistry. The results showed an
increase in p53 expression in the basal cell compartment of UVB-
exposed skin as compared to the non-UVB-exposed skin. However,
DSO has significant photoprotective effects by inhibition of damage
caused by UVB irradiation: a significantly lower fraction of cells
was p53 positive as compared to the non-DSO-treated skin. We
conclude that p53 expression is a sensitive parameter for the
detection of UVB-induced damage in the skin and suggest that DSO
could provide an efficient complement to photoprotective measures
and may contribute to reduce the DNA damage.
Keywords Date Seed Oil; Human Skin; Immunohistochemistry; p53
Address correspondence to Ines Dammak, Chez Rahmouni Ali,
faculté de droit, route Sidi Mansour Km 10. BP 704. 3000 Sfax,Tunisie.
E-mail: inesdammak@yahoo.fr
55
INTRODUCTION
Studies on the effects of ultraviolet (UV) light are very
important from the ozone depletion stand point. DNA is clearly
one of the key targets for UV light induced damage in a variety
of organisms [1]. The p53 protein is a transcription factor
which is upregulated in epidermal cells after UV irradiation.
UV induces high levels of p53 which then lead to the activation
of downstream genes, which subsequently induce cell-cycle
arrest, allowing DNA lesions to be repaired [2]. Thus, effective
photoprotection of DNA against harmful exposure to solar UV
is a critical issue. Some antioxidants, used as inhibitors of
apoptosis, can inhibit tumor initiation, tumor promotion, and
cell transformation [3]. Césarini et al. [4] investigated that
after the oral intake of an antioxidant complex “vitamins and
selenium” by healthy individuals, a reduction of the UV-induced
p53 expression was observed. Fruits of the date palm (Phoenix
dactylifera) are popular foods in many countries. Researchers
have found that date seed oil (DSO) has better oxidative stability
than most vegetable oils and also has a high antioxidant capacity
owing to its richness in polyphenol and tocopherol compounds
[5–8]. Recently, we have demonstrated that DSO prevents
hydrogen peroxide-induced oxidative stress in human skin organ
culture [9, 10]. In the current study, we have determined the
photoprotective effect of DSO to provide DNA protection, as
measured by means of p53 expression.
 56 I. DAMMAK ET AL
MATERIALS AND METHODS
Date Seed Oil Extract
Seeds of dates were soaked in water, washed and then dried
at about 50◦ C. Lipid extraction was carried out with an SER
148 solvent extractor [5]. The extraction was carried out over
a 30-min period, with thimbles immersed in boiling petroleum
ether, and 60 min of reflux washing. The solvents from seed
oils were removed under a stream of nitrogen and then stored
at −20◦ C. Part of DSO was dissolved in dimethyl sulfoxide
(DMSO) to obtain concentration of 100 µg/ml (stock solution).
Skin Samples
Connect Tissue Res Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 11/14/14
All skin samples (n = 12) were obtained from healthy adults
(25 to 40 years of age) undergoing abdominal plastic surgery.
These samples have the same skin color (brown) and phototype
(IV) “Fitzpatrick classification.” Ethical permission for this
study was obtained from the local Research Ethics Committee
and prior to initiation of the study, each subject was informed
about the purpose of the study and gave informed consent.
UVB Irradiation
UVB irradiation was delivered with a Biotronic device
(Vilber Lourmat, Marne la Vallée, France) equipped with
a dosimeter in which the UVB lamp emitted a continuous
For personal use only.
spectrum between 280 and 320 nm range and the peak emission
was recorded at 312 nm. The source of UVB radiation was a
band of 4 UVB lamps equipped with an electronic controller to
regulate UV dosage at a fixed distance of 24 cm from the lamps
to the surface of the culture plates.
Organ Culture
For at least 2 weeks prior to the experiment, areas were not
exposed to UV light (artificial or natural). The protected skin was
cut into pieces of ∼3 × 3 mm and placed dermal side down in
contact with DMEM (Dulbecco’s minimum Eagle’s medium)
complemented with 10% FCS, 200 µg/ml glutamine, and
penicillin/streptomycin (100 U/ml and 100 µg/ml respectively).
The explants were cultured in a humidified incubator containing
5% CO2 and 95% air.
Experimental Design
We prepared four different wells: control: (UVB = 0 mJ/cm2 ;
DSO = 0%), DSO alone (UVB = 0 mJ/cm2 ; DSO = 12%)
UVB alone (UVB = 200 mJ/cm2 ; DSO = 0%), and DSO +
UVB, (UVB = 200 mJ/cm2 ; DSO = 12%). Skin samples were
incubated with the selected concentration of DSO (12% of the
total volume of the culture medium) for 48 hr before UVB
exposure. This dose was selected in our previous studies because
it was an optimal concentration which gives the maximum pro-
tective effect [9]. After rinsing with PBS, skin explants in fresh
PBS were exposed to the selected dose of UVB (200 mJ/cm2 ).
This dose, equivalent to 1-hr sun exposure, was selected in
our preliminary studies because it was the optimal dose that
generated clear damage without succeeding a disastrous effect
in the skin structure in to evaluate thereafter the protective effect
of DSO (data not shown). Control biopsies were subjected to
the identical procedure but without exposure to UVB. After
rinsing with PBS, skin biopsies in fresh medium were placed
for 24 hr in an incubator. Thereafter, skin biopsies were fixed in
formalin (10%), embedded in paraffin, and sectioned into 4 µm
sections. These sections were used for immunohistochemical
staining.
Immunohistochemistry
After overnight incubation at 37◦ C, tissue sections were
deparaffinized in xylene 101 and rehydrated in ascending grades
of alcohol. Endogenous peroxidase activity was exhausted
by incubation of tissue sections in 0.3% H2 O2 for 30 min
at room temperature. Tissue sections were then treated with
DAKO retrieval solution (DAKO); 20% rabbit serum in
TBS was used for blocking. The monoclonal antibody DO-7
(DAKO) in 1:200 dilution in 2% rabbit serum was used
to stain p53. It was incubated with the samples overnight.
Biotinylated rabbit antimouse IgG (DAKO) was used as a
secondary antibody. All tissue sections were stained under
similar conditions to ensure equal staining quality. In the
negative control samples, the primary antibody was not
added.
Scoring of p53 Immunoreactivity
The level of p53 expression was evaluated according to the
staining intensity of 100 epidermal nuclei per biopsy, scored by
two independent observers. The results are presented as values
of p53 which were an average of three measures.
Statistical Analysis
The significance of the differences was determined by
student’s t-test; p ≤ 0.05 is considered significant. Results were
presented as means ± SD.
RESULTS
P53 immunoreactivity patterns of the biopsy specimens,
presented as the mean number ± SD of 100 examined cells,
are summarized in Table 1. The mean value of p53 expression
in the whole non-UVB exposed skin group, DSO alone, was (1 ±
1.2). It is evident from this table that the fraction of p53-positive
nuclei was significantly increased (22 ± 2.3; p < 0.05) in the
cells after UVB exposure, compared to non-UVB-exposed skin.
However, DSO provided high protection because the degree
of p53 expression significantly decreased (5 ± 1.1; p < 0.05)
compared to the non-DSO-treated skin.
Immunohistochemical staining for p53 in the epidermis of
human skin revealed a dispersed pattern of staining in diverse
tissue sections. The non-UVB exposed skin biopsies, DSO
 DATE SEED OIL INHIBITS p53 EXPRESSION 57

TABLE 1
Evaluation of p53 immunostaining reactions presented as the
mean number ± SD of 100 cells examined
Number of cells
Treated skin p53 immunostained
Controls 0
DSO alone 1 ± 1.2
UVB alone 22 ± 2.3
DSO + UVB 5 ± 1.1
DSO = date seed oil, UVB = ultraviolet.
Connect Tissue Res Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 11/14/14

alone, showed only weak p53 expression in a few scattered

cells in the basal layer compartment (Figure 1). However,
immunohistochemical analysis of biopsies irradiated with dose
of 200 mJ/cm2 of UVB show a definite increase in epidermal
p53 staining. Remarkable changes were noted in the epidermis
following exposure to UVB; a predominance of staining of basal
cells of the epidermis was seen as compared to the non-UVB-
FIG. 2. p53 immunoperoxidase detection in UVB-exposed skin, UVB alone
exposed skin (Figure 2). Evaluation of p53 expression in skin (magnification × 400).
before and after treatment with DSO revealed the tendency
of DSO to induce a decrease in the level of p53 expression.
p53 expression in the DSO-protected skin was similar to our event. It has been shown that p53 protein accumulation results
observations in control skin; a weakness of staining of basal in growth arrest via the induction of genes involved in cell
For personal use only.

cells of the epidermis was seen. It is evident that DSO provided
high protection concluded because p53 levels were remarkably
reduced (Figure 3).
DISCUSSION
Induction of DNA damage by UVB triggers many cellular
responses. Among these, p53 activation appears to be a critical
cycle control [2]. During cell cycle arrest, DNA repair can
occur. However, if there is too much damage, the cell undergoes
apoptosis [11]. Previous mechanisms proposed for the beneficial
effects of antioxidants have involved blocking apoptosis and
preventing of oxidative damage to cellular DNA [12]. In
the present study, we determined the photoprotective effect
of DSO to provide DNA protection, as measured by p53
expression.

FIG. 1. p53 immunoperoxidase detection in non-UVB-exposed skin, DSO FIG. 3. p53 immunoperoxidase detection in DSO-protected skin, DSO +
alone (magnification × 400). UVB (magnification × 400).
 58 I. DAMMAK ET AL
This study implicates reactive oxygen species (ROS) as
important intermediates in the UVB radiation-dependent in-
crease in p53 in human skin. The considerable enhancement
of the UVB radiation increase in p53 confirmed that p53 was
able to be elevated by a DNA damage-dependent pathway
[13]. As skin exposure of UVB radiation induces structural
changes in cellular DNA, the dietary botanical supplements
with antioxidant activity should be more effective against the
photodamaging effect of UVB radiation. Our results suggest
that DSO provided high protection as concluded from the fact
that p53 levels were significantly decreased.
Antioxidant and radical scavenging properties of DSO were
demonstrated in our previous studies [9, 10]. DSO pretreatment
Connect Tissue Res Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 11/14/14
was reported to prevent H2 O2 -induced oxidative stress in human
skin organ culture providing free-radical-scavenging effects [9,
10]. The DSO extract consisted of various substances with
different mechanisms of skin protection (e.g., hydroxytyrosol
and α-tocopherol), and the effectiveness of this mixture may
result from the synergism of their component actions [6]. The
biological properties of hydroxytyrosol, the main component
(10.21%) of the total polyphenols in DSO, are based on
scavenging of ROS [14]. Arief et al. [3] studied the effects
of hydroxytyrosol, the major antioxidant component present
in olive oil, on UVB-induced skin carcinogenesis in mice.
They revealed the tendency of hydroxytyrosol to induce a
For personal use only.
significant decrease in the level of p53 expression [3]. Alpha
(α)-tocopherol, the main component (24.97%) of the total
tocopherols in DSO, is effective at scavenging ROS in cell
membranes [15]. Vile et al. revealed that α-tocopherol inhibited
the p53 increase measured after ultraviolet A (UVA) irradiation
of cultured fibroblasts [15]. The combination of DSO active
components seems to modulate cellular pathways activated by
UVB exposition endowing with radicals scavenging ability.
Our data suggest that DSO could provide an efficient
complement to photoprotective measures and may contribute to
reduce DNA damages. Future studies applying flow cytometric
analysis of p53 expression in the epidermis could provide a more
accurate estimate of DSO effectiveness.
Declaration of Interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
REFERENCES
1. Eiichiro, M., Akihisa, T., Kou, K., Saki, K., Daiki, T., Megumi, U., Shoko,
N., Ken, O., Mitsuo, H., Norio, M., Masakatsu, W., Yoshiya, F., and Takeo,
O. (2008). Time course and spacial distribution of UV effects on human
skin in organ culture. J. Radiat. Res., 49, 269–277.
2. Pfeifer, GP., You, YH., and Besaratinia, A. (2005). Mutations induced by
ultraviolet light. Mutat. Res., 571, 19–31.
3. Arief, B., Nazim, U.A., An, W., Toshinori, B., Osamu, N., Toshihiko, O.,
Masato, U., and Masamitsu, I. (2000). Protective effect of topically applied
olive oil against photocarcinogenesis following UVB exposure of mice.
Carcinogenesis, 11, 2085–2090.
4. Césarini, JP., Michel, L., Maurette, JM., Adhoute, H., and Béjot, M. (2003).
Immediate effects of UV radiation on the skin: modification by an an-
tioxidant complex containing carotenoids. Photodermatol. Photoimmunol.
Photomed., 19, 182–189.
5. Besbes, S., Blecker, C., Deroanne, C., and Attia, H. (2004a). Date seeds:
chemical composition and characteristic profiles of the lipid fraction. Food.
Chem., 84, 577–584.
6. Besbes, S., Blecker, S., Deroanne, C., Bahloul, N., Lognay, G., Drira, NE.,
and Attia, H. (2004b). Date seed oil: phenolic, tocopherol and sterol profiles.
J. Food. Lip., 11, 251–256.
7. Besbes, S., Deroanne, C., Drira, NE., Bahloul, N., Lognay, G., Drira, NE.,
and Attia, H. (2004c). Quality characteristics and oxidative stability of date
seed oil during storage. Food. Sci. Techn. Inter., 5, 333–338.
8. Besbes, S., Blecker, C., Deroanne, C., Lognay, G., Drira, NE., and Attia,
H. (2005). Heating effects on some quality characteristics of date seed oil.
Food Chem., 91, 469–476.
9. Dammak, I., Ben Abdallah, F., Boudaya, S., Besbes, S., El Gaied, A., Attia,
H., and Hentati, B. (2007). Date seed oil limit oxidative injuries induced
by hydrogen peroxide in human skin organ culture. Biofactors., 29, 137–
145.
10. Dammak, I., Ben Abdallah, F., Boudaya, S., Besbes, S., El Gaied, A., Attia,
H., and Hentati, B. (2007). Effects of date seed oil on normal human skin
in vitro. Eur. J. Dermatol., 6, 516–519.
11. Davenport, V., Morris, JF., Motazed, R., and Chu, AC. (1999). p53 induction
in normal human skin in vitro following exposure to solar simulated and
UVB irradiation. J. Photochem. Photobiol. B., 49, 177–186.
12. Ming, L., Jifi, C., Pelling, J., Edward, C., and Douglas, E.B. (1998). An-
tioxidant action via p53-mediated apoptosis. Cancer. Res., 58, 1723–1729.
13. Courtois, SJ., Woodworth, CD., Degreef, H., and Garmyn, M. (1997). Early
ultraviolet B-induced G1 arrest and suppression of the malignant phenotype
by wild-type p53 in human squamous cell carcinoma cells. Exp. Cell. Res.,
233, 135–144.
14. D’Angeloa, S., Ingrossoa, D., Migliardia, V., Sorrentinoa, A., Don-
narummab, G., Baronic, A., Masellaa, L., Tufanob, M.A., Zappiaa, M.,
and Gallettia, P. (2005). Hydroxytyrosol, a natural antioxidant from olive
oil, prevents protein damage induced by long-wave ultraviolet radiation in
melanoma cells. Free Rad. Biol. Med., 7, 908–919.
15. Vile, GF., Tanew-Liitschew, A., and Tyrrell, RM. (1995). Activation of
NFkappaB in human skin fibroblasts by the oxidative stress generated by
UVA radiation. Photochem. Photobiol., 62, 463–468.