DOI:10.1111/j.1600-0625.2009.00865.x
www.blackwellpublishing.com/EXD
Original Article
Protective effects of a cream containing Dead Sea
minerals against UVB-induced stress in human skin
Meital Portugal-Cohen1, Yoram Soroka2, Zeevi Ma’or3,4, Miriam Oron3,4, Tamar Zioni3,4,
François Menahem Brégégère2, Rami Neuman5, Ron Kohen1 and Yoram Milner2
1
Department of Pharmaceutics, School of Pharmacy, the Hebrew University of Jerusalem, Jerusalem, Israel;
Department of Biological Chemistry, Silberman Institute of Life Sciences, the Hebrew University of Jerusalem, Jerusalem, Israel;
3
Ahava – Dead Sea Laboratories, Israel;
4
Dead Sea and Arava Science Center, Dead Sea, Israel;
5
Department of Cosmetic Surgery, Hadassa Hospital Ein Karem, Jerusalem, Israel
Correspondence: Prof. Yoram Milner, Head of the Myers Laboratory of Skin Biology and Biochemistry, Department of Biological Chemistry,
The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel, Tel.: +97226585051, Fax: +97226585428, e-mail: milner@vms.huji.ac.il.
2
Accepted for publication 27 January 2009
Abstract
Background: Dead Sea (DS) mud and water are known for their
unique composition of minerals, and for their therapeutic
properties on psoriasis and other inflammatory skin diseases.
Their mode of action, however, remains poorly known.
Objectives: To analyse the ability of Dermud
TM
, a leave-on skin
preparation containing DS mud and other ingredients like DS
water, zinc oxide, aloe-vera extract, pro-vitamin B5 and vitamin
E, to antagonize biological effects induced by UVB irradiation in
skin when topically applied in organ cultures.
Methods: We have used human skin organ cultures as a model to
assess the biological effects of UVB irradiation and of DermudTM
cream topical application. Skin pieces were analysed for
mitochondrial activity by MTT assay, for apoptosis by caspase 3
assay, for cytokine secretion by solid phase ELISA, for overall
antioxidant capacity by ferric reducing antioxidant power and
Oxygen radical absorbance capacity assays (epidermis) or by cyclic
voltammetry (external medium), and for uric acid (UA) content
by HPLC.
Results: We report that UVB irradiation decreases cell viability,
total antioxidant capacity and UA contents in the epidermis of
skin organ cultures, while increasing the levels of apoptosis in cells
and their cytokine secretion. Topical application of DermudTM
decreased all these effects significantly.
TM
has
protective, anti-oxidant and anti-inflammatory properties that can
antagonize biological effects of UVB irradiation in skin. It may
therefore be able to reduce skin photodamage and photoaging,
and more generally to reduce oxidative stress and inflammation in
skin pathologies.
Conclusions: Our results clearly show that Dermud
Keywords: apoptosis – Dead Sea mud – inflammatory cytokines –
reactive oxygen species (ROS) – UVB irradiation
Please cite this paper as: Protective effects of a cream containing Dead Sea minerals against UVB-induced stress in human skin. Experimental Dermatology
2009.
Introduction
Recent lifestyle evolution has boosted the development of
open-air leisure activities, and consequently increased the
levels of skin exposure to solar ultraviolet rays (UV) (1). This
phenomenon is believed to be determinant in the elevation
of skin cancer frequencies observed in the last decades (2,3).
UVC rays, defined by the 200–280 nm wavelength range,
are efficiently blocked by the atmosphere and do not reach
the skin. UVB (280–320 nm) affect the basal layer of epidermis, causing DNA lesions and protein damage (4–6).
UVA (320–400 nm) penetrate deeper and reach the dermis,
inducing endothelial cell necrosis, blood vessel damage and
collagen degradation (7–9).
ª 2009 John Wiley & Sons A/S, Experimental Dermatology
Skin irradiation by either UVA or UVB is believed
to affect biological tissues through initiation of photooxidative reactions, impairment of antioxidant balance
and increase of reactive oxygen species (ROS) cellular
levels. The activation of ROS-sensitive signalling pathways
(6,10) may actively participate in short- and long-term
skin pathologies like erythema, inflammation, photoaging
and skin tumors (11,12). Recent data suggest that sunscreens may not be so efficient to protect skin against
UV-induced cancers, failing to avoid long-term consequences of photodamage (13–17). Topically applied
substances able to modulate ROS-sensitive signalling
pathways induced by UV exposure might provide a
better protection, by interfering with UV-induced
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Portugal-Cohen et al.
biochemical pathways that actually cause pathologic or
undesired effects. Moreover, this protective role could be
extended to other skin disorders where the same pathways are involved.
The therapeutic and cosmetic properties of Dead Sea
(DS) mud and water have been well established, especially
when combined with irradiation by atmosphere-filtered
sun rays at low-altitude (18–21). Their unique composition
is specially rich in magnesium, calcium, sodium, potassium, zinc and strontium (22,23), some of which are
known to influence metabolism. For instance, topically
applied Mg2+ ions have an anti-inflammatory effect (24)
and accelerate skin barrier recovery in cooperation with
Ca2+ ions (25); zinc was shown to be involved in epidermis proliferation and wound healing (26). Underlying biological mechanisms, however, have not been elucidated,
and how hydrophilic salts can cross the hydrophobic barrier of stratum corneum (SC) and reach deep skin layers
to exert their effects, is one more question (27). Minimal
penetration through non-lipidic channels such as hair follicles and sweat ducts may occur, but its functional importance is under debate (28). Therapeutic efficacy of DS
minerals on psoriasis, contact and atopic dermatitis might
be enhanced by easier trans-epidermal penetration in
lesional areas, due to SC damage (29). Alternatively,
limited penetration may be enough to activate signalling
pathways by receptor binding (26,27), or even non-binding
activation may occur, as hypothesized by Speich and
Bousquet (30). In fact, we have observed that topical
application of DS salts on skin organ cultures, or skin in
vivo, can down-regulate some aging biomarkers and
stimulate mitochondrial activity (31).
Topical application of DS mud, known as ‘mud pack’, is
a classical procedure in DS climatotherapy and normally
requires final washing off with clear water. The originality
of DermudTM cream resides in its formulation as a leaveon-skin emulsion with a high content in DS mud and minerals, in combination with aloe-vera, pro-vitamin B5 and
vitamin E. DermudTM was patented as a pharmaceutical
composition by Ahava-Dead Sea Laboratories Ltd. (Ma’or
et al., 2000, US Patent No. 6582689). Preliminary results
from our laboratory revealed that DS mud and salts can
preserve epidermal cells against UV-induced photoaging
and inflammatory effects. In this work, we analyse the
capability of DermudTM to protect human skin against
UVB-induced biological effects when topically applied
in vitro.
Methods
Materials
Unless otherwise specified, fine chemicals were obtained
from Sigma-Aldrich, Steinheim, Germany.
2
Human skin organ culture model for biological
tests on epidermis
Skin fragments were obtained with informed consent from
20 to 60-years-old healthy women, who underwent breast
or abdomen reduction. Samples were cut into pieces of
approximately 0.5 · 0.5 cm and placed, dermal side down
and epidermal side up, in 35 mm diameter Petri dishes
containing DMEM (Dulbecco’s Modified Eagle’s Medium,
Biological Industries Beit Haemek, Israel) at 37C, under
5% CO2. DermudTM cream was applied on to the airexposed epidermis, 36 h before irradiation as described
below. The samples were incubated for another 0.5 h (oxidative stress), 24 h (epidermis viability, apoptosis level and
inflammatory cytokine secretion), or 48 h (epidermis viability). At the end of the postirradiation period, epidermis
was separated from dermis by 1-min heating in phosphate
buffered saline (PBS) at 56C. Remaining culture medium
was collected.
Skin exposure to UVB irradiation
Before irradiation, all culture medium was discarded and
the skin fragments were washed in PBS to remove all traces
of cream. Enough PBS was added to cover the dermis, and
the sample was irradiated with a UVB source (VL-6.M
lamp, emission spectrum 280–350 nm, emission peak
312 nm, filter size 145*48 mm, Vilber Lourmat, Torcy,
France) at 200 mJ ⁄ cm2. Immediately after irradiation, PBS
was replaced by DMEM growth medium, and skin was
further incubated for various periods of time.
Preparation of epidermal extracts
Epidermis was separated after heat treatment as described
above, then immediately deep-frozen in liquid nitrogen
and kept at )68C. Tissue homogenisation was carried out
in phosphate buffer (pH 7.2) on ice, using a mechanical
homogenizer (Polytorn PT MR 2100, Kinematica AG,
Lucern, Switzerland). The amount of epidermal proteins
in the extract was measured by the Bradford method
(32), using bovine serum albumin as a standard (Pierce,
Rockford, IL, USA). Samples were stored at )68C until
analysis.
Viability measurements through mitochondrial
assay
Epidermis viability was estimated using a simple colorimetric assay of mitochondrial activity. Skin slices were heated
and epidermis was separated as described above. Samples
were transferred into wells containing 200 ll of 0.5 mg ⁄ ml
(3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (‘‘MTT’’, Cat # 475989, Calbiochem, San Diego, CA,
USA), and incubated 1 h at 37C. The resulting, precipitated stain (formazan crystals) was extracted for 30 min at
room temperature in 0.5 ml isopropanol (Frutarom, Haifa,
ª 2009 John Wiley & Sons A/S, Experimental Dermatology
Cream with Dead Sea minerals inhibits UV stress in skin
Israel). Hundred microlitre aliquotes were transferred into
96-well plates, and optical density was measured in duplicate at 568 nm (33).
Apoptosis determination by caspase 3 assay
Epidermis samples were incubated in 100 ll PBS containing
2.5 lm Ac-DEVD-AMC as a substrate, with 0.02% Triton
X-100 and 10 mm DTT, at 37C in a 96-well plate (34). Fluorescence of the released coumarin derivative was measured
at 390 ⁄ 435 nm, using a Fluostar-BMG spectrofluorimeter
(Offenburg, Germany). Activity was given by the fluorescence-versus-time slope, calculated over 30 min in linear
range. Results were normalized with respect to MTT assays,
performed on each sample after the caspase 3 reaction.
Antioxidant capacity
Overall antioxidant scavenging capacity, i.e. ability to
donate electrons to ROS, was measured either in epidermis
or in external medium, using different methods.
Ferric reducing antioxidant power assay
This method, described by Benzie and Strain (35), consists
in measuring the ability of epidermal extracts to reduce ferric (Fe3+) ions by using a redox-linked colorimetric assay.
Briefly, Fe3+ ions form a complex with 2,4,6-tri-(2-pyridyl)1,3,5-triazine (TPTZ, Fluka, Buchs, Switzerland), which
turns intense blue when reduced to the ferrous form at low
pH. Ferric reducing antioxidant power (FRAP) reagent was
prepared by mixing 300 mm acetate buffer (pH 3.6), 10 mm
TPTZ (3.1 mg ⁄ ml in 40 mm HCl) and 20 mm FeCl3Æ6H2O
(5.4 mg ⁄ ml in H2O), in a ratio of 10:1:1. A total of 40 ll of
epidermal extracts were added to 300 ll of FRAP reagent,
and 150 ll aliquotes were transferred to a 96-well plate.
Absorbance was measured 10 min after mixing at 595 nm,
using a ‘PowerWave 340’ microplate spectrophotometer
(Bio-tek Instruments, Winooski, VT, USA). Phosphate buffer (homogenization solution) was used as blank. Results
were quantified by comparison with standard curves
obtained with a Trolox solution, and expressed in lmoles
Trolox per milligram protein. All measurements were performed at room temperature, sheltered from direct sunlight.
Oxygen radical absorbance capacity assay
Amounts of epidermal antioxidants were also measured
with
2,2¢-Azobis(2-amidino-propane)
dihydrochloride
(AAPH) as a peroxyl generator, using the Oxygen radical
absorbance capacity (ORAC) assay described by Benzie and
Strain (35), adapted to fluorescein labelling (36). Measurements were performed on a Fluostar Galaxy plate reader
(BMG, Offenburg, Germany) equilibrated at 37C, with
excitation and emission set up at 485 nm and 520 nm,
respectively. We used Trolox as a calibration standard. All
reagents were prepared in 75 mm phosphate buffer (pH
ª 2009 John Wiley & Sons A/S, Experimental Dermatology
7.4). A total of 40 ll aliquotes of epidermal extracts, Trolox
dilutions, or phosphate buffer as a blank, were transferred
into a 96-well microtiter plate. Fluorescein was added to a
final concentration of 96 nm. ORAC fluorescence was read
every 2 min for 70 min. Peroxyl radical-induced oxidation
started immediately after AAPH addition. Results were
quantified by comparison with Trolox as above. Total antioxidant capacity was calculated by measuring the area
below the kinetic curve.
Voltammetric assay of reducing power
The overall reducing power of extracellular media was
assayed using a cyclic voltammeter (Electrochemical Analyzer, CH Instruments, Austin, TX, USA). Culture media
samples were placed in a well with three electrodes: a glassy
carbon, working electrode of 3.3 mm diameter, an Ag ⁄ AgCl
reference electrode and a platinum wire as auxiliary electrode (37). Potential was applied to the working electrode
at a constant rate (100 mV ⁄ s). Reducing power was determined from the cyclic voltammogram and was composed
of two parameters: the peak potential (Ep(a)), which reflects
the ability of several compounds with close potentials to
donate electrons, and the anodic current (AC), which measures the concentrations of these compounds (35). The
working electrode was checked before each series of measurements, by performing a cyclic voltammogram of 1 mm
potassium ferricyanide in PBS.
Uric acid dosage
Although uric acid (UA) is a waste product derived from
purine metabolism, it has the ability to react with harmful
oxidants like peroxynitrite, and its consumption may therefore be an indicator of ongoing oxidation. We determined
UA levels in epidermis using a HPLC system (36) composed of a LC 320 pump (Kontron, San Diego, CA, USA),
a 20 ll injection loop, a reversed phased, 4 lm-pore,
250 · 4.6 mm C-18 column (Supleco, Bellefonte, PA, USA)
and a voltammetric detector. Skin extracts were thawed
and centrifuged at 14 000 rpm for 10 min at 4C, and
supernatants were collected for analysis. After filtration on
nylon filters (0.2 lm pores, 17 mm diameter, National Scientific, Rockwood, TN, USA) 25 ll aliquotes were injected
to the HPLC system. The mobile phase (100 mg ⁄ ml EDTA,
0.1 M Acetic acid buffer at pH 4.75, 1% Tetrabutylammonium hydroxide) was delivered at a flow rate of
0.8 ml ⁄ min. The voltage applied to the samples was
+600 mV, with a sensitivity of 50 nA. Uric acid concentration was determined by comparison with standard UA
solutions (Sigma Chemicals, St Louis, MO, USA).
Cytokine secretion
Skin organ culture media were collected after 24 h incubation in standardized conditions. Concentrations of IL-1a,
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Portugal-Cohen et al.
IL-6, IL-8, IL-10 and TNFa were measured by solid phase
ELISA (37) using highly sensitive immunoassay kits (Siemens Medical Solutions Diagnostics, Hounslow, UK).
Briefly, a polystyren microtiter plate was coated with a
cytokine-specific monoclonal antibody. Standards and samples were then placed in the wells and incubated with the
immobilized antibody. After immune binding of the antigen, unspecific compounds were washed away, and a second enzyme-linked, monoclonal or polyclonal antibody
directed to the same antigen, was added. After completion
of a ‘sandwich’ formation, unbound molecules were washed
away, and a colorogenic substrate was added to reveal the
enzyme-linked antibody. The enzymatic reaction was
stopped in linear phase, and assessed by colorimetry using
an Immulite 1000 analyzer (Siemens Healthcare Diagnostics, Germany). Cytokin concentrations were determined by
comparison with standard solutions.
Statistical analysis
Each experiment was performed at least in triplicate. Average values are given with standard error of the mean
(SEM). Differences between average values were tested for
significance using the unpaired Student t-test and considered as significant for P < 0.05.
Results
Figure 1. Epidermal cell viability and apoptosis upon UVB irradiation
and Dermud treatment. Human skin cultures were irradiated by UVB as
described in the Methods section, and the effects were measured 24 h
after irradiation. (a) Apoptosis was monitored in epidermis by
measuring the extent of caspase 3 activity in epidermal extracts, in
response to various UVB doses. (b) Cell viability was determined in
epidermis extracts by MTT assay, in response to various UVB doses. (c)
Organ cultures were treated with DermudTM for 36 h, then irradiated
with UVB at 200 mJ ⁄ cm2, and cell viability was evaluated by MTT assay
as before. (d) Caspase 3 activity was measured in epidermal extracts
after DermudTM treatment and UVB irradiation. Error bars represent
SEM. In (c) and (d), statistical significance of differences between
average measurements was evaluated by Student t-tests; P-values
appear at bottom-right of each diagram.
UVB dose range
UVB effects on cultured keratinocyte are optimally studied
at doses ranging between 5 and 50 mJ ⁄ cm2 (38,39). But living epidermis resides in deep layers efficiently protected by
the SC and requires harsher irradiation to be efficiently
challenged. Experimental doses commonly range from 150
to 250 mJ ⁄ cm2 (38,40). We determined that in our conditions, apoptotic caspase 3 activity in epidermis increased
linearly with UVB dose up to 300 mJ ⁄ cm2 (Fig. 1a), while
cell viability measured by MTT mitochondrial assay underwent a limited decrease, generally about 40% (Fig. 1b).
Hence we chose to use a single dose of 200 mJ ⁄ cm2 in all
experiments, which in fact corresponds to 15 min–4 h of
solar irradiation (41), a common period of time for outdoor exposure.
Reduction of UVB-induced cytotoxicity by
Dermud
treatment
Two kinds of tests were performed to evaluate the ability
of DermudTM to interfere with UVB-induced cell toxicity
in epidermis. First, mitochondrial activity was monitored
using the MTT test. UVB exposure caused a 20% decrease
48 h after irradiation, but previous application of DermudTM reversed this trend, with a 20% increase in the
same conditions (Fig. 1c). Second, the final stage of apoptosis was investigated by assaying caspase 3 activity in epi-
4
dermal extracts prepared 24 h after UVB irradiation. UVB
exposure caused a drastic 10-fold increase, but a previous
treatment with DermudTM reduced this increase by 80%
(Fig. 1d). These results demonstrate a profound effect of
DermudTM to curb UV-induced cytotoxicity in epidermal
cells.
Anti-oxidative effect of Dermud
Antioxidant potential of skin samples was assessed by three
different methods. FRAP was measured in epidermis
extracts, and displayed a significant 30% decrease after
UVB irradiation. Topical treatment by DermudTM
enhanced FRAP by 50% in non-irradiated controls, and
remarkably, this DermudTM-enhanced antioxidant power
was not affected by UV irradiation (Fig. 2a).
Oxygen radical absorbance capacity in epidermis, contrary to FRAP, was not significantly modified by UVB irradiation, in treated as well as in untreated samples. The two
methods, however, converged to show a significant increase
in skin antioxidant power after DermudTM treatment, in
both non-irradiated and irradiated samples (Fig. 2b).
The overall reducing power of culture media, which
reflects the amounts of low molecular weight antioxidants
(LMWA) released by skin samples, was measured using
ª 2009 John Wiley & Sons A/S, Experimental Dermatology
Cream with Dead Sea minerals inhibits UV stress in skin
Figure 2. Effect of DermudTM on epidermis reducing power and total
antioxidant capacity. Skin organ cultures were treated with DermudTM,
irradiated as above, and epidermis was collected 30 min after
irradiation. (a) Ferric reducing antioxidant power (FRAP) was determined
in epidermal extracts as described in the Methods section and
normalized to protein contents. (b) Total antioxidant capacity was
determined in epidermal extracts, using the oxygen radical absorbance
capacity assay as described in the Methods section and normalized to
protein contents. Error bars represent SEM. Statistical analysis as above.
cyclic voltammetry. Two anodic waves, corresponding to
two groups of LMWA with similar oxidation potentials,
were detected in all tested conditions, one at 619–668 mV
and the other at 720–890 mV. After UV exposure, the
exogenous reducing powers associated with these two waves
were significantly decreased, by 20% and 10%, respectively.
This effect disappeared when DermudTM was applied
before irradiation, as shown in Fig. 3.
Limitation of UVB-induced uric acid decrease
Uric acid concentration was measured in epidermal extracts
of skin organ cultures as described in the Methods section.
Fig. 4 shows a 30% decrease of UA levels after UVB irradiation, partially restored by DermudTM pretreatment.
Figure 3. Effect of DermudTM on extracellular medium reducing
power. Skin organ cultures were treated with DermudTM and irradiated
as above. 30 min after irradiation, the culture medium was collected
and its reducing power was determined by cyclic voltammetry, as
described in the Methods section. Two anodic waves were detected at
624–668 mV (a) and 716–859 mV (b). Anodic current measurements
were measured in DermudTM-treated and in untreated samples. Error
bars represent SEM. Statistical analysis as above.
Figure 4. Effect of DermudTM on epidermal uric acid. Skin organ
cultures were treated with DermudTM and irradiated as above.
Epidermis was collected 30 min after irradiation. UA levels were
determined in epidermal extracts by HPLC separation and voltammetric
detection, as described in the Methods section, in DermudTM-treated
and untreated samples. Error bars represent SEM. Statistical analysis as
above.
Limitation of UVB-induced cytokine secretion in
skin organ cultures
Discussion
We found that UVB irradiation increased the secretion of
inflammatory cytokines in skin samples in vitro: the
amounts of TNFa in culture medium were raised by a factor 5, while those of interleukins IL-1a, IL-6 and IL-8 were
significantly increased. DermudTM application prior to irradiation drastically inhibited this effect (Fig. 5). It also
decreased the basic levels of cytokin secretion in non-irradiated skin samples by 40% for IL-6 and IL-8, and by 70%
for IL-10, with IL-1a and TNFa remaining unchanged. As
a whole, these results suggest that DermudTM may have a
wide anti-inflammatory competence in skin tissue.
Skin exposure to UVB rays damages epidermal cells
enhances ROS generation and inflammatory processes and
leads to different skin pathologies (42,43). Therefore, topical application of anti-inflammatory and antioxidant agents
may reduce UVB-induced skin disorders. It has been
shown indeed that UVB-induced damage in skin cells can
be efficiently limited by plant-derived antioxidant compounds (38,44–47).
This study has been focused on DermudTM, a patented,
leave-on skin emulsion enriched with DS mineral mud, DS
water, zinc oxide (ZnO), a-tocopherol (vitamin E) and
ª 2009 John Wiley & Sons A/S, Experimental Dermatology
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Portugal-Cohen et al.
Figure 5. Effect of DermudTM on cytokine secretion by skin. Skin
organ cultures were treated with DermudTM and irradiated as above.
Culture media were collected 24 h later; their cytokine contents were
determined by ELISA as described in the Methods section and measured
in DermudTM-treated and untreated samples. (a) IL-1a; (b) IL-6; (c) IL-8;
(d) IL-10; (e) TNFa. Error bars represent SEM. Statistical analysis as
above.
panthenol (pro-vitamin B5). DS minerals are expected to
interfere with UV-induced inflammation and photoaging,
because of their anti-inflammatory and antipsoriatic
properties, that are commonly exploited in climatotherapeutic cure at the DS site (21). Moreover, DS minerals
were shown to down-regulate aging pathways in epidermal cells, presumably through intracellular signalling
(48).
Among other DermudTM components, ZnO is common
in dermal creams. It can absorb UV rays and behaves as an
antioxidant (49). However, direct smearing of DermudTM
on the probe of a UV light meter (Lutron UV-340, Taiwan) did not change UVB intensity measurements (Ma’or
Z, unpublished results). Hence, we can exclude the possibility that DermudTM could work as a UV filter. Vitamin E
in topical applications can protect skin against UV effects
by increasing the levels of small antioxidant molecules (50),
increasing ROS-scavenging activities (50,51), reducing lipid
peroxidation (51,52) and preventing DNA photodamage
(53). Panthenol is converted into panthotenic acid when
topically applied on skin and can reduce experimental
ultraviolet-induced erythema (54). It is also common in
cosmetic preparations due to its emollient, anti-inflammatory and cell-regenerative properties. The whole set of
DermudTM constituents is therefore expected to cooperate
in a protective anti-oxidant and anti-inflammatory effect in
UVB-exposed skin.
6
UVB rays target epidermal DNA and produce pyrimidine
dimers, resulting in activation of p53 transcription factor,
excision repair and ⁄ or apoptosis (55). Fos and Jun are activated and AP-1-dependent transcription is induced;
matrix-degrading metalloproteinases (MMPs) are upregulated and collagen synthesis is inhibited. While this DNAdependent response occurs several hours after irradiation, a
DNA-independent pathway, mediated by ROS derived from
extranuclear chromophores, can be detected earlier and
with lower UVB doses (56,57). It activates EGF receptor,
ERK, JNK and p38 MAP kinases, stimulates c-Jun synthesis
and activates AP-1-dependent transcription (58). DNAdependent and independent pathways cooperate to induce
MMPs and inhibit collagen synthesis, thereby harming dermal extracellular matrix and causing photoaging symptoms.
As DermudTM is not a UVB filter, it cannot prevent direct
photodamage. But because it combines antioxidant potency
with anti-inflammatory properties, it is expected to antagonize the ROS-derived, early UVB response, and possibly
also MAP kinase-dependent steps induced by DNA
damage, all of them leading to photoaging.
We applied DermudTM cream topically on human skin
organ cultures, then proceeded to UVB irradiation and
analysed the effects on epidermis. Measurements of mitochondrial activity and of UVB-induced apoptosis by caspase 3 assay converged to show that DermudTM efficiently
inhibits UVB-mediated cytotoxicity. As UVB-induced
apoptosis is considered as a vital process to eliminate
potential tumor cells, however (4,59), the possibility that
anti-cytotoxic properties of DermudTM might facilitate the
persistence of UV-induced carcinogenic mutations has to
be considered. This question is in fact relevant to antioxidants in general, as was discussed for green tea extracts in
particular (46). We can argue that improved vitality is
expected to stimulate the DNA-repair process and decrease
the mutation rate, and common experience has indeed led
to consider antioxidants as anticarcinogens (60). Yet, this
issue has to be evaluated for each product.
We showed previously by voltammetric experiments that
DermudTM has antioxidant potency [Ep(a) 1000 mV; AC
10 lA], so that its ability to enhance mitochondrial activity and to limit apoptosis is likely to proceed by neutralization of UVB-induced ROS excesses. Using two different
methods (FRAP and ORAC), we observed that DermudTM
treatment increases the total antioxidant power of epidermis, in both irradiated and unirradiated samples. Remarkably, UVB irradiation does not affect DermudTM-enhanced
antioxidant power, showing that UVB-derived ROS are
completely neutralized by DermudTM. As the cream
does not penetrate through the SC, this can mean that
most UVB-induced ROS may originate from surface
chromophores located in upper epidermal layers. Alternatively, DermudTM could prevent intrinsic epidermal ROS
ª 2009 John Wiley & Sons A/S, Experimental Dermatology
Cream with Dead Sea minerals inhibits UV stress in skin
production through signal transduction, in accordance with
reports that external ion concentrations can modulate cellular ROS levels (61). The fact that FRAP values decrease
after UVB irradiation, while ORAC is not significantly
affected can be explained by differences between the two
methods: the FRAP assay titrates only Fe3+ reducing
reagents, while the ORAC assay monitors all antioxidant
molecules that is able to react with peroxyl radicals.
Consistently with FRAP and ORAC experiments, the
antioxidant power of external medium, corresponding to
low molecular weight antioxidants (LMWA) secreted by
skin, was decreased by UVB irradiation in untreated, but
not in DermudTM-treated samples. This further suggests
that UVB-derived, LMWA-consuming ROS, were totally
neutralized by DermudTM. The slight decrease observed in
non-irradiated samples upon DermudTM treatment may be
explained by an independent effect of DermudTM to limit
LMWA secretion.
Uric acid is one of the LMWAs (62). It is an example of
adaptation to oxidative stress, being originally a waste
product derived from hypoxanthine and xanthine oxidation
by xanthine oxidase and xanthine oxidoreductase (63). UA
is a scavenger of peroxynitrite, a harmful and versatile oxidant derived from interaction between nitric oxide and
superoxide radicals (64–66). UVB irradiation reduces epidermal UA content, presumably through increased consumption by peroxynitrite and derivatives. Hence, the fact
that DermudTM prevents UA depletion indicates a possible
role in peroxynitrite scavenging.
UVB irradiation stimulates the expression of nuclear
factor-jB (NF-jB) (67), as do other pro-oxidants or ROSregulated factors (68,69). NF-jB activation is an early event
in inflammatory response, which targets TNFa, IL-1a, IL-6
and IL-8 genes (70,71). Thus, NF-jB makes a link between
ROS and inflammatory cytokine expression. Our results
provide further evidence that UV irradiation induces these
cytokines, as well as anti-inflammatory IL-10. Although
paradoxical, the simultaneous induction of IL-10 and proinflammatory cytokines has already been reported (70,72).
Topical application of DermudTM drastically reduces the
secretion of these cytokines following UV irradiation,
thereby limiting keratinocyte contribution to UVB-induced
inflammatory response. A similar inhibition of IL-6, IL-8
and IL-10 secretion was found in unirradiated samples,
suggesting that DermudTM may also control the basal level
of inflammation in skin.
In conclusion, we have demonstrated that DermudTM
cream possesses antioxidant, anti-apoptotic and antiinflammatory properties that reduce harmful effects of
UVB involved in photoaging. Further studies are needed to
elucidate the underlying mechanisms and to determine
individual contributions of its constituents.
ª 2009 John Wiley & Sons A/S, Experimental Dermatology
Acknowledgements
This work was funded by the Dead Sea & Arava Science Center, Israel (to
RN and YM), and by the David and Ines Myers Fund of Cleveland, Ohio,
USA (to YM).
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