DERMATOLOGY

CARINE NIZARD1*, EMMANUELLE LEBLANC-NOBLESSE1, MILENE JUAN1, TONY XU2, SYLVIANNE SCHNEBERT1
*Corresponding author
1. LVMH Recherche. Life Science Department, 185 Avenue de Verdun, 45800. Saint Jean de Braye. France.
2. LVMH Recherche. Asia Innovation Center. 3511, Tower 1, Plaza 66, No.1266 Nanjing road, Shanghai,
200040, China.

Carine Nizard

Pollution skin protection

by cosmetic formula against placebo:
ex-vivo model with 32 pollutants

KEYWORDS: pollution, skin morphology integrity scoring, MDA, skin explant

Abstract The skin, is an interface between the body and the surrounding atmosphere and is therefore the primary
contact for ambient pollutants. Once inside the skin, the pollutants can accumulate and form free radicals
that generate DNA and protein damage thus causing serious premature aging. It has been demonstrated that oxidized proteins in the
skin accumulate with age and that they can be eliminated by proteasome in cytosol, and Lon protease in mitochondria. We
previously developed two active ingredients (Phaeodactylum tricornutum and Hibiscus sabdariffa extracts) which activate these two
systems in order to eliminate oxidized proteins in these two cellular compartments. Here, we show that a skin explant, exposed to 32
pollutants (27 heavy metals and 5 hydrocarbons) under a patch can be a good ex-vivo model for pollution damage and that our
formula is a good protector against pollution damage (skin morphology integrity scoring) and lipids peroxidation (by
Malondialdehyde measurement).

INTRODUCTION issue, so we need to counteract adverse effects of oxidized

For decades, extrinsic skin aging has been known to result
from chronic exposure to solar radiation and, more recently
to pollution (1). Pollution is a world-wide issue and the skin is
the body’s first line of defence against these stressors. It’s not
necessary to inhale polluted air for it to harm the skin as many
topically applied substances get absorbed through the skin
into the bloodstream and tissues of the body (2). A major
mechanism by which ambient PM (particulate matter) exerts
its detrimental effects is through the generation of oxidative
stress which is an important contributor to extrinsic skin
aging. Particles in the nanosize range, especially those from
road traffic, are considered to be among the most harmful
components of ambient PM. These nanoparticles cause
oxidative stress partly because of their physical properties, i.e.
small size but large surface per unit mass, making them highly
reactive toward biological surfaces and structures. Pollutants
bind to the stratum corneum, penetrate into the epidermis,
or become metabolized. In this process the structural and
functional integrity of the epidermal barrier seems to be
directly damaged at the sites of contact or impaired through
indirect pathways, such as increased inflammation. Pollutants
could also have a direct impact on development and
aggravation of atopic dermatitis and which is known to be
related to skin barrier defects (3).
Free radicals, DNA and protein damage cause serious
premature aging (4). As oxidative stress induces the
accumulation of oxidized proteins in the skin, it is a serious
protein accumulation in the skin with appropriate cosmetic
solutions.
It has been demonstrated that oxidized protein accumulation
in the skin can be eliminated by proteasome in cytosol, and
Lon protease in mitochondria. We previously developed
two active ingredients, Phaeodactylum Tricornutum (5) and
Hibiscus Sabdariffa extracts (6) which activate these two
systems in order to eliminate oxidized proteins in these two skin
cellular compartments.
Here, we set up an ex-vivo experiment, on skin explants,
to measure the adverse effects of a model of pollution (27
heavy metals and 5 hydrocarbons) on skin integrity and MDA
(malondialdehyde) which is involved in the accumulation of
oxidized proteins. We performed assays with a placebo as
well as active ingredients of cosmetic formulas to measure
their putative protection effect.
ExPERIMENTAL SECTION
Material
Cosmetic formula contains Phaeodactylum Tricornutum
extract (Soliance, France) and Hibiscus Sabdariffa flower
extract (Greentech. France) plus excipients. Placebo
contains only excipients.
Pollution is deposited on patch applied on explants and is a
mix of Certi Pur® solution (Merck) containing 27 heavy metals
plus 5 hydrocarbons.

26 H&PC Today - Household and Personal Care Today, Vol. 10 nr. 1 January/February 2015
Heavy metals the MDA is extracted with butanol (liquid / liquid), which
AS 0.01mg/mL, B 0.001mg/mL, Ba 0.001mg/mL, Be 0.0005mg/ allows us to measure the MDA by spectrofluorimetry.
mL, Ca 0.005mg/mL, Cd 0.001mg/mL, Cr 0.001mg/mL, Cu
0.001mg/mL, Fe 0.001mg/mL, Hg 0.0025mg/mL , K 0.0495mg/
mL , Li 0.001mg/mL , Mg 0.0005mg/mL ,Mn 0.0005mg/mL, Na RESULTS AND DISCUSSION
0.01mg/mL, Ni 0.0025mg/mL, P 0 .005mg/mLg, Pb 0.01mg/mL,
Sc 0.0005mg/mL, Sa 0.01mg/mL, Sr 0.0005mg/mL Te 0.01mg/ Histology
mL , Ti 0.001mg/mL, Y 0.0005mg/mL, Zn 0.001mg/mL. (ICP Histological studies were made at Day 5. Photos were
multi-element standard V, Merck, Germany). taken (figure 1) and histological scoring of epidermis was
made (Figure 2).
Hydrocarbons
Benzene, 2μL/mL, Anthracene : 1 mg/mL, Naphtol : 1mg/mL,
Xylene 2μL/mL, Toluene 2μL/mL (Sigma, France)

Methods

Ex-Vivo experiments
12 mm diameter explants were prepared from a 36 year old
woman abdominoplasty and placed in a survival medium at
37°C in a humid atmosphere enriched with 5 percent CO2. (7
for detailed protocol).
Daily (from Day 0 to Day 4), cosmetics formulas (either
containing or not containing the two active ingredients)
were applied on explants. When no formulation is applied,
we named it control. On Day 4, a few hours after the
application of the two different formulas, a mixture of heavy
metals and hydrocarbons was deposited on a filter paper
disc and applied on explants. The entire survival medium
was also renewed on day 4. On day 5, explants were taken
Figure 1. Masson’s Trichrome staining of Day 5 explants. (A) Control;
and survival medium was removed and frozen at -20°C.
(B) pollution treatment; (C) Complete formula and pollution
After 24 hours in buffered formalin, samples were dried and treatment; (D) Placebo formula and pollution treatment
impregnated in paraffin 5 micron sections were made and
mounted on slides for histological studies. 3 explants by
condition were prepared.

Histology
For morphology observation, the paraffin sections were
stained with Masson’s trichrome Goldner variant. The general
morphology was evaluated by microscopic examination
(Leica optical microscope type DMLB at the magnification of
40). 5 photos by explants were taken.
Epidermis morphology was evaluated by scoring scales:
number of cell layers, cell morphology and cellular
alterations. The scores for each parameter were rated and
compiled to calculate a score from 0 to 100, reflecting the
Series1
general epidermal morphology. 5 scores were given to
each explant (one by photo) by a trained expert: 15 scores
were obtained for the 3 explants by condition. Mean and Figure 2. Histologic scoring of epidermis structure of Day 5 explant
confidence interval were calculated for each condition and
student test performed.

MDA Assay At day 5 on the control group, the general morphology is

The malondialdehyde (MDA) is a product of lipid peroxidation
of cell membranes. Free radicals induced by oxidative
stress (UVA, pollutants, heavy metals, pesticides ...) degrade
polyunsaturated lipids and generate hydroperoxides which
decompose into radical intermediates and aldehydes whose
main representative is MDA (8).
MDA was determined in the survival medium on day 5 by
the TBARS method (thio barbituric acid reactive species)
modified to render it more sensitive and specific. The TBARS
assay is based on the reaction of MDA with thiobarbituric acid
to give a coloured compound which is then determined by
spectrophotometry. To increase the specificity of the assay,
similar to that observed in the day 0 batch. The effect of the
different formulas is compared with relation to pollution.
Treatment of explants with mixed heavy metals plus
hydrocarbon induced a markedly altered morphology which
validates the experimental procedure (-70 percent epidermis
scorage p<0,0001).
The result when comparing the effect of the application of
the complete formula containing active ingredients and the
placebo formula is different on the general morphology when
compared to the control batch.
The complete formula presents a good level (p<0,0001) of

H&PC Today - household and Personal Care today, Vol. 10 nr. 1 January/February 2015 27
protection on the general skin morphology and particularly
on epidermis morphology (score of the epidermis: 60 versus
30). The placebo formula presents only a slight but statistically
significative (p<0,05) protection of the general morphology
and the epidermis morphology (score: 40 versus 30).
As formula is applied on explants before the patch with
pollutants, we don’t have proofs that 100 percent of
pollutants reach the skin. Formula could act as a physical
barrier against pollutants. We measure this protection with the
results of placebo which is non negligeable (score of 40). The
score of 60 of the formula is though the addition of protection
of the formula itself plus the effect of the ingredients.
MDA Assay
24 hours after treatment with heavy metals and hydrocarbons
(pollution), the concentration of MDA in the medium was
measured for all conditions. (Figure 3).
Control
Series1
Figure 3. MDA measurement
At Day 5, pollution induces an increase (+602 percent, p< 0,001)
of MDA level. Compared to the control treated with heavy metals
and hydrocarbons, the complete formula induces a significant
decrease of 22 percent** (p< 0,001) of MDA concentration in
the medium. The placebo formula induces a non-significant
decrease of the concentration of MDA in the survival medium.
Skin aging is the result of different mechanisms. One of the main
agents responsible for extrinsic aging is the reactive oxygen
species (ROS). Due to its interface function between the body
and the environment, the skin is chronically exposed to both
endogenous and environmental pro-oxidant agents, leading
to the generation of a variety of harmful substances. These free
radicals and ROS are generated from endogenous sources, such
as enzyme activity or activated neutrophils, and also external pro-
oxidant stimuli, such as pollution.
Reactive carbonyl species are small molecular weight carbonyls
that are activated by alpha,beta-unsaturation (such as
4-hydroxynonenal and acrolein), and beta-oxo-substitution (such
as malondialdehyde).
Reactive carbonyl species are especially dangerous because
they are by-products of cellular metabolism, including lipid
peroxidation, glycation, auto-oxidation of sugars, etc… Among
the damage caused by RCS, we find DNA damage, proteasome
degradation as well as cellular and extracellular protein alteration.
This last effect has been linked to skin deterioration which is seen in
our experiment with pollutants.
The two ingredients (Phaeodactylum Tricornutum extract and
Hibiscus Sabdariffa flower extract) have positive actions on
proteasome and Lon protease activities which in fine, decreases
28 H&PC Today - household and Personal Care today, Vol. 10 nr. 1 January/February 2015
the amount of altered proteins. These activities seem to have a
positive impact on skin protection (epidermal scoring and MDA)
after pollutants application. The exact composition of these two
extracts is not completely known and they may both contain
polyphenols, which could have an effect on MDA levels (9) or skin
barrier function (10). Hibiscus Sabdariffa flower extract is known to
have wound healing properties (11) which could explain, at least
in part, the effect of formula protection from pollutants.
Proteins highly modified by 4-hydroxy-2-nonenal that have
been found to accumulate both during skin aging and
upon UV irradiation of cultured human keratinocytes (12,13)
were previously shown to act as inhibitors of the proteasome
(14). Therefore, as observed for the extract from the algae
Phaeodactylum Tricornutum after UV irradiation (5) reducing the
occurrence of such oxidatively modified proteins by stimulating
the proteasome is expected to help preserve its critical function
both during aging and under oxidative stress conditions such as
pollution.
CONCLUSION
Interestingly, our ingredients from our formula are able to
significantly protect skin morphology and integrity from pollution
particules. Moreover, they significantly decrease MDA index.
Taken together, these results indicate that our product is able to
protect the skin from environmental pollution.
As we don’t have clear mechanistic clues for pollution skin
impairments, we should conduct further in-vitro and ex-vivo
experiments to decipher pollution pathways and how our active
ingredients act on putative actor’s pathways.
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The author has requested enhancement of the downloaded file. All in-text references underlined in blue are linked to publications on ResearchGate.