Evaluation of the impact of urban pollution on the quality of skin: A multicentre study in Mexico

Abstract

The impact of pollution on skin quality is becoming a very important subject, especially in big cities where industrial activities bring the most part of known pollutants. In the past years, a lot of publications reported potential explanations for pollutants impact on skin aging, including or not participation of UV or tobacco exposition. Thiele et al [2] for example described the lack of vitamin E under exposition to ozone. Sticozzi et al [3] evaluated the effect of some pollutants and their consequences on skin aging, with a focus on young or elderly subjects. More Recently, Vierkötter, et al [4] reported on the difference between intrinsic and extrinsic skin aging, taking into account specific approach of ethnicity. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.

Full text

Evaluation of the impact of urban pollution on the quality of skin: a
multicentre study in Mexico
M.-A. Lefebvre*, D.-M. Pham
†
, B. Boussouira
†
, D. Bernard
‡
, C. Camus
†
and Q.-L. Nguyen
†
*L’Oreal Research & Innovation, 11-13 rue Dora Mar, 93400 Saint-Ouen,
†
L’Oreal Research & Innovation, 188 rue Paul Hochart BP 553, 94152
Chevilly Larue, and
‡
L’Oreal Research & Innovation, 1, avenue Eug
ene Schueller, BP22, 93601 Aulnay Sous-Bois, France
Received 12 November 2014, Accepted 24 January 2015
Keywords: atmospheric pollution, chemical analysis, multicenter study, skin physiology/structure
Synopsis
OBJECTIVE: After pilot and preliminary studies aimed at identify-
ing pertinent biochemical parameters, a multicenter clinical study
was performed to evaluate the effect of pollution on human skin.
METHODS: The clinical study was performed in collaboration with
the ‘Centre R
egional de lutte contre le cancer de Montpellier’ and
the ‘National Institute of Public Health of Mexico’ on 96 subjects
in Mexico City (exposed to pollution) and 93 subjects in Cuerna-
vaca (less exposed to pollution). Both biochemical and clinical skin
parameters were studied.
RESULTS: The study demonstrated significant quantitative and
qualitative modifications of parameters related to sebum excretion
in Mexico City compared to Cuernavaca one: An increased level of
sebum excretion rate, a lower level of vitamin E and squalene in
sebum, an increase of lactic acid and a higher erythematous index
on the face of the subjects. In the stratum corneum, a significant
higher level of carbonylated proteins and a lower level of IL 1a
were noticed, as well as a decrease of ATP concentration with a
decrease of chymotrysin like activity, without modifications of cor-
neodesmosin content and trypsin like activity. From a clinical point
of view, a higher frequency of atopic and urticarial skins, a higher
frequency of red dermographism, an important seborrheic status at
the forehead level and a lower level of dandruffs were noted in
Mexico City population. The analysis taking into account the sex
does not modify the observed results.
CONCLUSION: The study demonstrated an important impact of
polluted environmental conditions on skin quality, evidencing
important modifications of superficial biochemical parameters. The
cause/effects relationships of these modifications remain, however,
to be further assessed by a complementary in vitro/in vivo
approaches.
R
esum
e
OBJECTIFS: L’effet de la pollution sur la peau humaine a 
et
e

evalu
eend

efinissant au cours d’une 
etude pilote, suivie d’une

etude pr
eliminaire, les param
etres biochimiques superficiels
impact
es, et en conduisant une 
etude clinique multicentrique.
M
ETHODES: L’
etude clinique a 
et
e conduite en collaboration avec
le ‘Centre R
egional de Lutte contre le Cancer de Montpellier’ et
‘l’Institut National de la Sant
e du Mexique’, sur 96 sujets de la ville
de Mexico (Zone expos
ee 
a la pollution) et 93 sujets 
a Cuernavaca
(zone moins expos
ee 
a la pollution). Des mesures biochimiques et
des observations cliniques ont 
et
e effectu
ees.
R
ESULTATS: L’
etude a d
emontr
e des modifications statistiquement
significatives des param
etres li
es 
a la production quantitative et
qualitative de s
ebum dans la ville de Mexico comparativement 
a
Cuernavaca : une augmentation de la vitesse d’excr
etion du
s
ebum, une concentration inf
erieure de vitamine E et de squal
ene
dans le s
ebum, une augmentation de l’acide lactique et un index

eryth
emateux sup
erieur sur le visage des sujets. Dans le stratum
corneum, un niveau sup
erieur de prot
eines carbonyl
ees, un niveau
inf
erieur de IL1a, une diminution de la concentration en ATP ainsi
qu’une diminution de l’activit
e ‘chymotrypsin like’ sans modifica-
tion de la corneodesmosine ou de l’activit
e ‘Trypsin like’. Sur le
plan clinique une fr
equence plus 
elev
ee de peau atopique ou
pr
esentant des caract
eristique d’urticaire, une plus grande
fr
equence de dermographisme et un niveau inf
erieur de pellicules
ont 
et
e not
es dans la ville de Mexico. L’analyse des r
esultats en
prenant en compte l’influence du sexe ne modifie pas les conclu-
sions tir
ees de l’
etude.
CONCLUSION: L’
etude d
emontre un impact important des condi-
tions environnementales d’exposition sur la qualit
e de la peau en
mettant en 
evidence d’importantes modifications des param
etres
biochimiques superficiels de la peau. Les relations cause/effets de
ces modifications restent cependant 
a^
etre 
evalu
ees au cours de
futures approches in vivo/in vitro.
Introduction
The impact of pollution on skin quality is becoming a very impor-
tant subject, especially in big cities where industrial activities
bring the most part of known pollutants. In the past years, many
publications reported potential explanations for pollutants impact
on skin ageing, including or not participation of UV or tobacco
exposition [1]. Thiele et al. [2] for example described the lack of
vitamin E under exposition to ozone. Sticozzi et al. [3] evaluated
the effect of some pollutants and their consequences on skin age-
ing, with a focus on young or elderly subjects. More recently,
Vierk€
otter, et al. [4] reported on the difference between intrinsic
and extrinsic skin ageing, taking into account specific approach of
ethnicity [5].
More specifically, Xu et al. [6] described the impact of ozone
(O
3
) on skin disorders, whereas Choi et al. [7] investigated in
the influence of dust on gene expression in human epidermal
Correspondence: Dang Man Pham, L’Oreal Research & Innovation, 188
rue Paul Hochart, BP 553, 94152 Chevilly Larue, France. Tel.:
+33 625754067; e-mail: dmpham@rd.loreal.com
©2015 Society of Cosmetic Scientists and the Soci
et
e Franc
ßaise de Cosm
etologie 1
International Journal of Cosmetic Science, 2015, 1–10 doi: 10.1111/ics.12203

keratinocytes (HEK) and reported on a significant increase in
cytochrome P450 1A1 (CYP1A1), CYP1A2 and CYP1B1 gene
expression, likely related to aryl hydrocarbon receptor (AhR) acti-
vation. A link between O
3
impact and AhR activation was also
described [8].
Considering the possible relationship between some pollutants
and the skin ageing process, the involvement of proinflammatory
response was also described [9], whereas Fortino et al. [10] demon-
strated the ability of certain environmental pollutants to affect ECM
turnover through modulation of specific matrix metalloproteases
(MMPs) and confirmed the higher susceptibility of old subjects to
exogenous pro-oxidant insults.
Of note, O
3
, which is considered as one of the main pollutants,
was also reported to interfere in an age-dependent manner with
wound-healing processes [11]. Moreover, skin exposure to high lev-
els of O
3
affected antioxidant levels and oxidation markers in the
stratum corneum (SC) and induced stress responses most likely by
indirect mechanisms [1].
Although pollutants’ induced SC modifications were scarcely
described, concomitant exposure to low doses of UV and O
3
was
shown to cause additive oxidative stress in SC and, in particular, a
depletion in vitamin E [12,13].
Although most of the above-listed pollutant’s effects were
described in mice, L’Oreal Research Group engaged, for quite a
long time already, to evidence pollution effects on human skin
quality, by crossing both in vitro and in vivo evaluation methods
generally based on biophysical or biochemical parameters.
A first approach included a preliminary study on one subject
exposed to pollution on half his face and a pilot study involving
two groups of eight subjects in two different places near Paris, one
exposed to intra muros Paris pollution and one less exposed (Neau-
phle le Chateau). More recently, a larger study was designed to
confirm the initial biophysical and biochemical data and to collect
possibly correlated clinical data. This multicentre study was per-
formed in Mexico and designed in collaboration with the ‘Centre
Regional de Lutte contre le Cancer’ CRLC, of Montpellier and the
‘Institut National de Sant
e Publique du Mexique’ INSP.
Materials and methods
Clinical and instrumental evaluation
One hundred and eighty-nine healthy volunteers were enrolled,
aged between 18 and 60 years. A first group included 96 volun-
teers living in the south-west district of Mexico City, particularly
subjected to high level of pollution. A second group included 93 vol-
unteers, living in Cuernavaca (500 000 inhabitants), a city located
50 km far from Mexico City, and considered as poorly exposed to
urban pollution. Mexicans usually consider Cuernavaca as the town
of ‘Eternal spring’, and the subjects have been recruited in north
part of the town, which is even much less exposed to pollution.
Volunteers belong to active and non-active categories of popula-
tion, and exclusion criteria were related to cigarette consumption
higher than 10 per day and to alcoholism. The volunteers signed a
written and informed consent defined by the local ethical commit-
tee. Demographic data are given in Table I.
The study was performed in two sequential steps from 7 Septem-
ber 1999 to 5 January 2000 in Cuernavaca (mean tempera-
ture =15.8 2.05°C) and from 3 February to 23 March 2000 in
Mexico City (mean temperature =16.9 3°C).
Instrumental evaluations (Fig. 1) were carried out by two INSP
members. Skin pH and moisturizing level were measured on the
forehead (three zones, using devices). Close to these areas, sebum
production was evaluated (casual level and sebum excretion rate –
SER). Erythematous and melanin indexes were measured on fore-
head, cheek and cheekbone using a Mexameter
â
(Courage and
Kashaka, K€
oln, Germany). Sampling (cotton pad and Dsquam
TM
,
Westwood Pharma.Inc., Buffalo, NY, USA)) was performed on spe-
cific location of the face and kept frozen (20°C) until analytical
evaluations.
A questionnaire including a series of question related to skin,
scalp and state was filled by a dermatologist. A clinical diagnosis
was also performed particularly focusing on skin evaluation, former
eczemas, seborrhoea dermatitis, rosacea events and usual cosmetic
routines.
Table I Demographic data of volunteers having participated to the study
Cohortes description Cuernavaca (North) Mexico (Town)
Duration 7 September 1999–5 January 2000 3 February 2000–23 March 2000
Average temperature 15.8 2°C 16.9 3°C
Total panel Number 93 96
Average age 33 15 years 31 15 years
Male panel Number 36 40
Average age 31 15 years 30 12 years
Female panel Number 57 56
Average age 34 13 years 32 15 years
Ethnic origin Hispanic 99% 97%
Complexion Fair 17% 1%
Dark 80% 94%
Very dark skin <3% <3%
Phototype III 85% 93.80%
IV
Hair color at 20 youngest Light brown 3% 17%
Dark chesnut 96% 80%
Black
Eyes color Coffee >91% >91%
2©2015 Society of Cosmetic Scientists and the Soci
et
e Franc
ßaise de Cosm
etologie
International Journal of Cosmetic Science, 1–10
Impact of urban pollution on the quality of skin M. A. Lefebvre et al.

Biochemical analysis
Biochemical analyses were carried out on samples collected on the
face of the subjects. Sebum was harvested using a cotton pad filled
with alcohol. Stripping of superficial stratum corneum was
obtained using corneodiscs D’Squam, at four specific location of the
face (Fig. 1).
Tocopherol, squalene, lactic acid and cholesterol were extracted
from the cotton pad using 5 mL of methanol, then filtered on
RC15 before analysis.
Squalene evaluation
Squalene analysis was performed with Perkin Elmer HPLC (Ville-
bon-sur-Yvette, France), with a diode array detector. Chromato-
graphic condition was an isocratic mode (H
3
CN 75%/isopropanol
12.5%/CHCl
3
12.5%) using a Beckman high-speed C8 column.
Vitamin E evaluation
Vitamin E was measured using a reverse-phase chromatographic
method from the same sample than for squalene evaluation. The
analysis was conducted on a Series 200 HPLC device from Perkin
Elmer (Series 200) equipped with a DECADE electrochemical detec-
tor (Antec, Zoeterwoude, Netherlands). The separation was made
using a SGE Exsil ODS2 chromatographic column, with a mobile
phase (MeOH 95.5%/CH
3
COOH 0.5%/LiClO
4
1gL
1
per H
2
O 4%),
in the isocratic mode.
Lactic acid determination
Lactic acid was determined using a colourimetric method with a
measurement kit from Sigma (ref. 735-10 St Quentin-Fallavier,
France). Lactic acid was transformed in pyruvate and hydrogen
peroxide, using lactate oxidase. Hydrogen peroxide was then used
to catalyse the oxidation of a coloured precursor, to give a colour
detected at 540 nm. The increase in the absorbance at the wave-
length was proportional to the lactic acid concentration. Concen-
tration of lactic acid was given in p.p.m. (lgmL
1
).
Cholesterol measurement
The cholesterol was measured using gas chromatography with squa-
lene as internal standard, on a 3400 GC device from Varian (Les Ulis,
France), equipped with a 8200CX injector. Cholesterol was separated
from the other compounds using an Alltech (Fisher Scientific, Ill-
kirch, France) EC-5 (SE54) column (30 m; 0.32 mm; 0.25 lm), with
Helium as the carrier gas. Concentration was given in lgmL
1
.
Interleukin IL1a
After extraction from the corneodiscs in 700 lL of PBS buffer, over-
night freezing at 80°C and thawing, Interleukin-1awas measured
by ELISA, according to the supplier’s recommendations (Amersham,
Les Ulis, France Kit Interleukin-1ahuman, ELISA system).
Adenosine triphosphate
Adenosine triphosphate (ATP) value was assessed on Dsquam Cor-
neodisc, sampled on the face, using a chemiluminescence technique
(Kit ATP Ref 1 699 709/Assay Kit HSII; Boehringer Manheim,
Roche Diagnostics, Meylan, France). Luminescence was produced
using a D-luciferine/lucipherase system, emitted photons were
quantified using a dedicated camera (CCD C2400-77), and imager
process was performed with an acquisition software ARGUS 50/MP
(K.K. Hamamatsu Photonics). Dsquams were treated in a Petri dish
with the adequate reactive (1.35 mL) and the emitted light signal
acquired during 8 min. ATP contained in sample was quantified
using a standard curve.
Corneodesmosin and oxidized proteins determination
These parameters were assessed by electrophoresis after extraction
and denaturation of proteins on corneodiscs samples. The corneodiscs
were collected in 12-well microtitre plate and lysed with 0.5 mL of a
Laemmli buffer (0.0625 mM Tris pH 6.8, 200 mM DTT, 2% SDS,
10% glycerol). After gentle shaking for 20 min at room temperature,
20 lL of supernatant was taken and frozen at 20°C until used for
quantification of carbonylated proteins. Then, the whole microtitre
plate was put at 60°C for 30 min, and the total extract was homoge-
nized using a potter device for 30 min, then centrifuged at 12000 G.
The supernatant was collected and frozen at 20°C until used for
detection of corneodesmosin and quantification of keratins.
Oxidized protein (8 lL samples) was separated by 1D 11% poly-
acrylamide gel electrophoresis. After coomassie blue staining, kera-
tins bands were quantified using the Bio-Rad (Marnes-La-Coquette,
France) Quantity One software. Oxidized proteins were revealed
and quantified using an Oxyblot (Chemicon, Temecula, CA, USA)
detection kit as described by Thiele [2]. The carbonyl groups of pro-
teins reacted with 2,4-dinitrophenylhydrazin, to give 2,4-dini-
trophenylhydrazon. The modified proteins were then separated by
electrophoresis, followed by a Western blot. The oxidized proteins
were directly quantified using the Quantity One software. Results
were expressed as cntmm
2
per ODumm
2
.
Corneodesmosin was quantified after electrophoresis and Wes-
tern blot using G36-19 antibody diluted 1/12500 (generous gift of
Dr. G. Serre) and ECL detection (Amersham). Corneodesmosin was
observed as a major band at 46 kDa, directly measurable with the
Quantity One software (Bio-Rad). Values were expressed the same
way than oxidized proteins.
Trypsin-like et chymotrypsin-like activity measurement
Corneodiscs were individually put in microtitre wells (12-well unit)
containing 500 lL phosphate buffer 50 mM (pH 7.0, 150 mM
NaCl, 0.1% TX100, 5 mM EDTA). After shaking for 24 h at 4°C,
Blenderm T6H
ATP T6H
ATP To
Sebumeter
+
pH
pH TopH T6H
Cotton sample
Lipids & vitamin E
Figure 1 Sampling of the subjects participating to the study.
©2015 Society of Cosmetic Scientists and the Soci
et
e Franc
ßaise de Cosm
etologie 3
International Journal of Cosmetic Science, 1–10
Impact of urban pollution on the quality of skin M. A. Lefebvre et al.

the total extract was collected and centrifuged for 10 min at
14 000 g. The supernatant was frozen at 20°C until analysis.
Total protein content was measured to further normalize the
enzymatic activities. Assay was run in 96-well microtitre plate.
After adding 150 lL of Pierce reactive to 20 lL samples and a 30-
min incubation at 37°C, absorbance at 540 nm was recorded.
Results, expressed as microgram protein per well, allowed the nor-
malization of the two enzymatic activities.
The trypsin and chymotrypsin-like activities were measured in
96-well microtitre plates. Twenty microlitre sample was mixed with
50 lL of either 5 mM chromogenic S2288 (trypsin like) or S2586
(chymotrypsin like) (Instrumentation Lab., Le-Pr
e-St-Gervais,
France), and 150 lL of a Tris/HCl 0.1 M pH 8 buffer and then
incubated for 40 h at 37°C. Absorbance at 405 nm was recorded
and concentrations given as DDO/lg of protein.
Oxidized protein measurement
Oxidized proteins were measured using an ELISA proprietary
method. A 500-lL fraction of corneodisc protein extract (see IL1 a
measurement protocol) was incubated at room temperature for
1.5 h with 100 lL 2,4-dinitrophenyl hydrazine (DNPH) reactive.
After coating of proteins on NUNC plates (Immuno Maxisorp
TM
;
Fisher Scientific, Illkirch, France), and exposing the sites by incuba-
tion with Tween 20 in PBS, samples were reacted with anti-DNP-
biotinylated rabbit antibody for 1 h at 37°C, then with streptavi-
din-biotinylated horseradish peroxidase complex (incubation 1 h at
37°C) followed by a 5-step washing using PBS solution. The final
reaction was started by addition of the o-phenylenediamine dihy-
drochloride reactive solution, followed by a 25-min incubation in
the dark and then stopped by addition of 2.5 mM H
2
SO
4
solution.
Quantification was performed by spectrophotometry at 490 nm.
Carbonylated residues were evaluated as oxidized BSA equivalent.
Statistical protocol
Statistical study has been carried out in parallel by the ‘Centre
R
egional de lutte contre le cancer de Montpellier’ and the Statisti-
cal service of L’Oreal Research & Innovation in Chevilly. The inter-
pretation of the results was performed by two-side Student t-test,
except when the result of the normality test of Shapiro–Wilk was
inferior to 1% for at least one of the two groups. In this case, statis-
tical analysis was performed using the nonparametric Mann–Whit-
ney U-test. Some significant parameters were graphically visualized
by the box-and-whisker plots (using median and with error bars
represent the confidence intervals level at 95%).
Results
Description of the groups in Mexico and Cuernavaca
The study included 36 men and 57 women (mean age
34 13 years) in Cuernavaca and 40 men and 56 women (mean
age 32 15 years) in Mexico City.
The mean exposition to O
3
and nitrogen dioxide (NO
2
) in Mexico
is known to be very high, higher than 0.25 and 0.2 p.p.m., respec-
tively. From the Mexican Ministry of Environment, 7–14% and
70% of the time is considered as ‘bad for the human health’ or
‘not satisfying for the human health’, respectively. On the contrary,
Cuernavaca, 60 km from Mexico, is considered as a town preserved
from pollution.
These two groups were mainly Hispanic. Some differences were
observed for the skin colour between the two groups (only 17.2%
and 1% of volunteers have lighter skin tone in Cuernavaca and in
Mexico City, respectively). Deep complexion was rare, lower than
3% in both groups. The skin phototype was in its vast majority
equal to III or IV, according to Fitzpatrick classification [14]. The
subjects used to wash their face mainly with water and, in some
cases, with soap.
Metrology data
The main results of the study are presented in Table II for biophysi-
cal parameters and in Table III for biochemical parameters. Bio-
chemical parameters significantly different between Mexico City
Table II Comparison of biophysical parameters
Metrological datas Cuernavaca (North) Mexico (Town)
Statistic test SignificativityDatas Area nmin
Mean
*Median Max nmin
Mean
*Median Max
Corneometric Forehead Lateral 92 30 59.2 101 96 21 50.8 88 Student P<0.001
Central 32 58.6 103 28 53.2 126 P<0.004
Cutaneaous pH Forehead Lateral 92 4.2 *5.1 6.8 96 4.1 *5.2 6.8 Mann–Whitney NS
Central 3.4 *5.15 7.1 3.9 *5.3 6.6 NS
Sebumetric casual level Forehead Lateral 92 14 *175 282 96 3 *155 205 Mann–Whitney NS
Central 52 *216.5 286 3 *199 249 NS
SER
Sebum excretion rate
Forehead Lateral 67 3 *64 205 96 23 *100 208 Mann–Whitney P<0.001
Central 3 *86 240 15 *119.5 247 P<0.002
Melanic index
Mexametry
Forehead 75 354 *541 623 51 472 *539 590 Mann–Whitney NS
Cheekbones 471 *519 610 485 *526 600 NS
Cheeks 465 *527 679 480 *530 612 NS
Erythematic index
Mexametry
Forehead 75 524 630 689 51 591 636.7 690 Student NS
Cheekbones 535 626 648 570 634.7 589 NS
Cheeks 538 627 679 573 628.4 672 P=0.03
4©2015 Society of Cosmetic Scientists and the Soci
et
e Franc
ßaise de Cosm
etologie
International Journal of Cosmetic Science, 1–10
Impact of urban pollution on the quality of skin M. A. Lefebvre et al.

and Cuernavaca are detailed in Fig. 2. Moisturizing, measured by
corneometry, was significantly higher in Cuernavaca population
(P<0.001 on central zone), indicating a dryer skin in Mexico City
population. Although cutaneous pH mean values were equivalent
in the two populations, a difference between SCL and SER values
was evidenced. The SCL values were not different whatever the
sample zones, but the SER values were significantly different
(P<0.001) for the central zone and for the lateral zone
(P=0.002).
Using mexameter for both erythematous and melanin indexes,
no significant difference was observed, except for the cheeks ery-
thematous index (P=0.003).
As for the composition of sebum, important differences were
observed between the two populations. Vitamin E and squalene
concentrations were significantly (P<0.001) lower in Mexico City
population, whereas lactic acid concentration appeared as signifi-
cantly higher (P<0.001). On the contrary, cholesterol values did
not differ between the two populations. Nevertheless, the ratio
between squalene and cholesterol was significantly different
(P<0.001).
Considering the biological parameters, the results for chymotryp-
sine-like activity (SCCE/KLK7) were significantly (P<0.001)
higher in Cuenavaca population, whereas no difference was
observed for trypsin-like activity (SCTE/KLK5). Oxidized proteins
were found higher in Mexico City population than in Cuernavaca
population. Interleukin 1a, as ATP values were significantly higher
in Cuernavaca population (P<0.05, P<0.001, respectively).
Clinical evaluation by dermatologists
The results of the clinical evaluation are summarized in Table IV.
A first evaluation demonstrated that analysis of the values,
obtained along volunteers’ questionnaires and quantified by the
dermatologists, did not evidence significant differences by sex popu-
lation, except for family antecedents of sensitive skin, found higher
in Mexico City population. Globally, there were more dermogra-
phism and urticarial antecedents in Mexico City population. Der-
matologists did not evidence significant differences between the two
populations as for cutaneous signs of sun aggression, acne signs,
psoriasis or other diseases signs. Although scalp problems were
found higher in Mexico, a special impact of dandruff was noted in
Cuernavaca population. No clinical evidence was evidenced, with
regard to erythematous scalp, folliculitis disease, psoriasis, other
scalp disease, face skin disease or dry skin.
A clinical evidence of greasy skin was noticed for nasal and peri-
nasal zones and found higher in Mexico City population, whereas
for Cuernavaca population, cheeks were found greasier. Finally, no
difference was found for squamous skin, facial erythematous skin
and fine skin.
Discussion
The influence of pollutants on the skin quality is a major subject,
especially in the big industrial areas all over the world. Pollutants
are very diverse, from some air (O
3
,NO
2
,...) components of the
air to industrial residues (polycyclic aromatic hydrocarbon, nano-
particles, ...). What stays as a question is the relative amount of
all these pollutants in the daily environmental exposition to the
human. A way to address this question is to follow the official
index in the dedicated zone or to measure directly the pollutants,
using trapping devices and adequate analytical determination.
This approach has been tested previously to this study in the
L’Oreal Group along a preliminary approach in Paris City
(Table V). According to Table III; 24 September 1998 was consid-
ered on a 1–14 scale, as a day with a pollution index of 4 on a
1–14 scale.
Samples were taken on a person exposed for this very day to
urban pollutants, including tee-shirt pieces and teenax samples (Su-
pelco) weared by the subject. Using a specific gas chromatographic
methodology, a large number of pollutants were identified in the
air (carbonylated substances and in particular acrolein, volatile
compounds such as benzene, toluene, xylene, some halogenated
compounds such as chlorofluorocarbon substances (Freon 3 and
5), linear, branched, saturated or non-saturated hydrocarbons,
some alcohols and ketons, substituted benzenic substances with
ethyl, diethyl, triethyl, propyl groups, benzaldehyde and phenyl
Table III Comparison of biochemical parameters
Biophysical/Biochemical parameters Unit
Cuernavaca
(North) Mexico (Town) Statistic test of Mann–
Whitney Cuernavaca VS
Mexico significance
threshold of 0.05nMedian nMedian
Ratio vitamin E/squalen pg lg
1
SQ 93 391 96 36.2 Very significant P<0.001
Cholesterol concentration lgmL
1
, extracted solution 92 6.39 92 6.7 No significant
Squalen concentration lgmL
1
, extracted solution 93 77.6 96 42.7 Very significant P<0.001
Lactic acid concentration lgmL
1
, extracted solution 92 10.6 96 22.9 Very significant P<0.001
Oxidized protein (Western blot) Arbitrary unit: cntmm
2
per ODumm
2
92 2720 95 6875 Significant P<0.05
Corneodesmosin value Arbitrary unit: cntmm
2
per ODumm
2
92 4223 95 3949 No significant
Trypsin like activity (SCTE) DDO(405 nm) per lg protein 92 4.98 95 4.68 No significant
Chymotrypsin like activity (SCCE) DDO(405 nm) per lg protein 92 1.19 95 0.77 Very significant P<0.001
Interleukin 1apg lg
1
protein 86 101 91 81.1 Significant P<0.05
ATP value pM per desquam 87 128 96 48.3 Very significant P<0.001
Ratio squalen/cholesterol lglg
1
92 11.7 92 7.6 Very significant P<0.001
ATP, adenosine triphosphate.
©2015 Society of Cosmetic Scientists and the Soci
et
e Franc
ßaise de Cosm
etologie 5
International Journal of Cosmetic Science, 1–10
Impact of urban pollution on the quality of skin M. A. Lefebvre et al.

ethyl acetate). Of note, no pollutants were detected in the tee-shirt
sample at the limit of detection of the GC/MS device.
From this first study of pollution and pollutants that confirms
the observation from Finlayson-Pitts et al. [15], it could be con-
cluded that pollution and pollutants are very diverse and that it
was very difficult to measure the exact concentration of the differ-
ent pollutants. From an analytical point of view, this diversity was
difficult to address completely, and the nature and amounts of
these pollutants could significantly vary from zone to zone.
It was then concluded that in this type of study, it was more
important to compare a polluted zone to a less polluted zone instead
of precisely measuring the pollutants environment. In the case of
this Mexico study, Cuernavaca was selected as the ‘less polluted
zone’ and Mexico City as the polluted zone. We used many well-
known methods to evaluate biophysical and biochemical parame-
ters, already known as performance markers of skin cosmetics.
Results given in Tables II and III demonstrated that several bio-
physical and biochemical parameters were significantly different in
Cuernavaca and Mexico City zones, indicating that the way of life
and the nature of the environmental conditions (UV exposition,
pollution and nature of pollutants) could have affect skin quality.
Sebum secretion modifications
The following parameters have been obtained from sebum collected
at different zones of the face. Although the sebum casual level on
the central and lateral zone of the forehead was not different, the
sebum excretion rate (SER) was significantly different between Cu-
ernavaca and Mexico City, with mean value 64 and 100, respec-
tively (P<0.001). A similar difference (84 and 119.5,
respectively) was observed for the lateral zone of the forehead
(P=0.002).
Figure 2 Statistical evaluation of some biochemical parameter s in the two populations.
6©2015 Society of Cosmetic Scientists and the Soci
et
e Franc
ßaise de Cosm
etologie
International Journal of Cosmetic Science, 1–10
Impact of urban pollution on the quality of skin M. A. Lefebvre et al.

In the previous studies conducted internally, we evidenced a ten-
dency of the cutaneous pH to decrease when exposed to bad envi-
ronmental conditions which is not the case in this study where the
pH values observed in the two populations are considered as non-
different. Although the pH values are non-different for the two pop-
ulations, the lactic acid value was significantly (P<0.001)
increased in Mexico City (22.9) compared to Cuernavaca (10.6).
These parameters could indicate that skin was submitted to pollu-
tant-related oxidative stress, as also indicated by squalene and
vitamin E levels, known as faithful oxidative stress markers [16–
20]. The values found both for squalene and for vitamin E are par-
ticularly interesting. Known as a powerful endogenous antioxidant,
the squalene value in Mexico City (mean value 42.7) is signifi-
cantly lower than in Cuernavaca (mean value 76.6) with a high
level of significance (P<0.001). The same observation is made for
the exogenous antioxidant vitamin E with mean values equal to
36.2 in Mexico City compared to 391.1 in Cuernavaca (Significant
P<0.001). This observation confirms already published results
[21–23].
In normal conditions, squalene is associated with sebum excre-
tion and low values of the two parameters are normally related to
dry skin. Nevertheless, squalene and vitamin E are the main an-
tioxidants at the surface of the skin, and the decrease values indi-
cate that they are mobilized to correct oxidative conditions. The
corneometer values and the clinical evaluation seem to confirm
these observations.
Cholesterol has also been evaluated in this study. Cholesterol
has an epidermic origin, and the fact that the ratio squalene/
cholesterol is lower in Mexico City compared to Cuernavaca
indicates that the oxidative stress reaches mainly surface
lipids. As previously described, in humans, oxidized surface lipids
are viewed as potent inflammatory mediators in many skin
afflictions such as pityriasis versicolour or seborrhoeic dermatitis
[24].
Table IV Results of clinical evaluation
Clinical signs evaluated by dermatologists Difference between the two populations S/N
Difference between the
two populations (sex analysis) S/NS
Clinical evidence of cutaneous diseases
Atopy No
Antecedent of atopy Yes (>Cuernavaca) >Women
Antecedent of atopic eczema or others eczemas Yes (>Mexico City) Yes (>Mexico City)
Family Antecedent of sensitive skin No NS Yes (>Mexico City) Men analysis S
Tendency to redness/to take colour easily No No
Presence of acne rosacea No No
Presence telangiectasy No No
Presence of hand dermatite >in Women NS Yes (Women) NS
Presence dermographism >Mexico City for red dermographism NS >Mexico City for red dermographism NS
Antecedent urticaria >Mexico City S >Mexico City S
Antecedent allergy and intolerance No No
Evidence of cutaneous signs of sun aggression No No
Presence of acne signs on face No No
Presence of psoriasis on the body No No
Presence of other disease signs No No
Problems of hair scalp Yes Dandruffs >Cuernavaca VS Yes Dandruffs >Cuenavaca VS
Clinical Evidence of scalp disease
Clinical evidence of dandruffs Yes in Men S Yes in men S
clinical evidence erythematous scalp No No
Clinical evidence of folliculite disease No No
Clinical evidence of greasy scalp Yes men/women NS
Clinical evidence of psoriasis No No
Clinical evidence of other scalp disease No No
Clinical evidence of face skin disease
Clinical evidence of dry skin No No
Clinical evidence of greasy skin Yes (nasal and perinasal zones) >Mexico City S Yes (nasal and perinasal zones) >Mexico City
Yes (Cheek) >Cuernavaca S Yes (Cheek) >Cuernavaca ~S
Clinical evidence of squamous skin No No
Clinical evidence of facial erythematous skin No No
Clinical evidence of fine skin No No
Table V Mean values of the main air pollutants in Paris (France) on 24
September 1998 (Airparif organization)
Pollutants in Paris on
the 24 September 1998
Mean value (one to
nine measurement sites)
Ozone (lgm
3
)28
Carbon dioxide (p.p.m.) 398
PM10 (lgm
3
)39
Sulphur dioxide (lgm
3
)10
Nitrogen oxide (lgm
3
)30
Nitrogen dioxide (lgm
3
)75
Carbon oxide (p.p.m.) 8.6
©2015 Society of Cosmetic Scientists and the Soci
et
e Franc
ßaise de Cosm
etologie 7
International Journal of Cosmetic Science, 1–10
Impact of urban pollution on the quality of skin M. A. Lefebvre et al.

Cutaneous structure modifications
The following parameters have been obtained from stripping sam-
ples collected at the different zones of the face. The first parameter
that appears as modified concerns the forehead central zones mois-
turization with values equal, respectively, to 58.60 and 53.17, for
Cuernavaca and Mexico City, respectively, that are significantly dif-
ferent (P<0.001). Some values in Mexico are considered as very
low (values of corneometer <40). On a clinical point of view, the
same was observed for forehead lateral zone but with somewhat
less significance (P=0.004). This confirms the results obtained by
Wolf et al. [25].
By the past, Goldstein [26] described the enzymatic activities as
a response to environmental chemicals. Desquamation of the stra-
tum corneum arises mainly by proteolytic digestion of the desmo-
somal proteins. Two different serine proteases, the trypsin-like
protease (SCTE, Kalikrein 5, KLK5) and the chymotrypsin-like pro-
tease (SCCE, Kalikrein 7, KLK7), have been identified as main
actors in of this process [27]. Indeed, Caubet et al. [28] showed
that amongst the Kallikrein family, the (SCCE/KLK7/hK7) and
(SCTE/KLK5/hK5) were both involved into the desquamation pro-
cess with specific impact. In this study, SCTE/KLK5 and SSCE/
KLK7 were measured, and although SCTE/KLK5 mean values were
similar in both city samples (4.98 in Cuernavaca and 4.68 in Mex-
ico), SCCE/KLK7 activities were significantly different (1.19 in Cu-
ernavaca, 0.77 in Mexico City) (P<0.001). The origin of these
differences would deserve further investigation.
Finally, as a consequence of oxidative stress and the decrease in
endogenous or exogenous antioxidative defence, the value of oxi-
dized proteins is significantly higher (P<0.05) in Mexico popula-
tion (mean value 6875.4) than in Cuernavaca population (mean
value 2720.1). This result has to be related to other published
results [29–33].
Moreover, the ATP value in Mexico City population was signifi-
cantly lower (mean value 48.3) than in Cuernavaca (mean value
127.8) (P<0.001). Such ATP depletion could also be interpreted
as the results of increased oxidative stress, as described by Tiwari
et al. [34].
Interleukin 1 alpha, (IL-1a) is a primarily known as a major
pro-inflammatory cytokine. It is released from macrophages in
response to a diversity of stimuli and is identified as a major factor
in the many inflammatory conditions [10,35–39]. Besides, IL-1a
was also identified as a potential regulator of fibroblast proliferation
[10,20,35,37,40] and may play a role in SC formation and barrier
function homeostasis [17,41]. The significantly lower IL-1avalues
found in Mexico City than in Cuernavaca samples (81.1 and 100.8
respectively, P<0.005) might indicate some pollution-related
alteration barrier function. The significance of IL-1adecrease in the
most polluted area of the study needs however further investigation
because McCarty [42] described in normal human keratinocytes
(NHK) an increase in IL 1a, under ozone exposition.
Clinical evaluation
Even if some biochemical parameters seemed to be modified in the
two populations, in relation to the environmental conditions, did
these modifications correlate with the clinical evaluation made by
skilled dermatologists? From Tables II and IV, a good correlation
appeared between greasy skin status in nasal and perinasal zones
and sebum excretion rate.Considering antecedents of skin urticar-
ial or atopy diseases, their occurrence was higher in Mexico City
than in Cuernavaca population. As for skin quality, dermatologists
observe a tendency for more hand dermatitis and dermographism
in Mexico City. Unexpectedly, however, dandruff incidence was
found higher in Cuernavaca than in Mexico City. This might be
linked to a higher sebum casual level in Cuernavaca population,
but this remains to be further confirmed.
Globally, there was tendency to find more skin problems in the
more polluted zone and the relation between biochemical parame-
ters and clinical evaluation of skin quality could be established.
What remain to be established, however, are the mechanisms that
could explain these observations and in particular the role of the
biochemical factors controlling skin homeostasis. This would
require in-depth fundamental studies of skin deep layers’ reactions
to pollutants.
Conclusion
This study participated to the long-term L’Oreal’s quest of causative
links between pollution/environmental factors and skin quality. A
first pilot study having demonstrated that some skin biochemical
factors were modified in subjects placed in a more polluted area,
the present comparative study in Mexico City and Cuernavaca was
designed to confirm on a larger scale that the pollution was
involved in skin quality alteration. The second goal was to estab-
lish possible correlations between the evolution of these parameters
and the clinical evaluation conducted by skilled dermatologists.
Although studies were performed at similar mean temperature,
but at two different periods of the year, on populations with a
slight difference in phototype ratios (see Table I), we believe that
despite these possible weaknesses, the main influencing parameter
was pollution. We confirmed in this study the pollution-related
modification of skin surface parameters with a focus on quantita-
tive and qualitative sebum secretion. In particular, vitamin E is
the main antioxidant present at the surface of the skin. The values
of lactic acid have been found higher in Mexico population evi-
dencing impact of bad environmental conditions on skin cell
homeostasis.
Some skin biochemical parameters were also modified such as
oxidized proteins, SCCE/KLK7 activity, IL 1aand ATP contents. It
appeared that the impact of bad environmental conditions on the
skin was related mainly to oxidative stress. The dermatologist eval-
uation on a large scale confirmed that some clinical parameters
could be related to biochemical modifications. Nevertheless, further
work remains necessary to understand, on a fundamental level, the
relation between parameters observed in our studies and the in-
depth functioning of the different skin layers, when exposed to pol-
luted environmental conditions.
Acknowledgements
The authors would like to thank Dr. G. Sore for coordinating the
writing of the manuscript and Dr. B.A. Bernard for helpful discus-
sions and critical reading of the manuscript. The authors would
also like to thank people for their invaluable help at designing and
performing this study: Dr. P. Catroux, Mariette Gerber research
director at CRLC/INSERM (Montpellier) –Mauricio Hernandez sci-
entific director at INSP (Cuernavaca), Dr. Christiane Montastier sci-
entific director at l’Oreal. This study was totally funded by L’Oreal
Research and Innovation.
8©2015 Society of Cosmetic Scientists and the Soci
et
e Franc
ßaise de Cosm
etologie
International Journal of Cosmetic Science, 1–10
Impact of urban pollution on the quality of skin M. A. Lefebvre et al.

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et
e Franc
ßaise de Cosm
etologie
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Impact of urban pollution on the quality of skin M. A. Lefebvre et al.