Skin model of pollutants penetration helps demonstrate the protective activity of a natural water
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Presented by: Valerie Cenizo
The increase of air pollution has a major impact on human health and skin makes no exception. Environmental air pollutants are generated by the industrial activity, the combustion of fuel, but can also come from indoor pollutants like paints, glues and cigarette smoke. The exposition of skin to air pollutants has been associated with serious effects such as skin cancer, as well as inflammatory and allergic skin conditions. The skin permeability barrier prevents penetration of chemicals from the environment. It is ensured firstly by the lipids in the stratum corneum and secondly by the tight junctions. But large amounts of airborne pollutants can overwhelm the skin’s protective abilities and even alter the barrier function, accelerating pollutants penetration. It is therefore necessary to develop protective shields for the skin and active ingredients that help strengthen skin’s natural defense.
Réotier water (RW) is a calcium-rich natural water from the French Alps, which has been used since ancient times to cure and soothe skin conditions. We previously showed on reconstructed human epidermis that RW could reinforce the permeability barrier by inducing tight junctions and cornified envelope proteins expression and limited the penetration of the Lucifer yellow (Cenizo V. IFSCC 2018).
To test whether RW was able to prevent the pollutants penetration, we setup a skin model of pollutants penetration. In a proprietary sterile device made of a hermetic cell, the skin explant was placed on a receptor compartment containing culture medium, allowing the culture for several days. We sprayed RW daily on the skin explants and measured the Trans-Epidermal Electrical Resistance (TEER) at different time points for 7 days. After only 5 hours, TEER had already increased by 30% (p<0.0001) in treated skin compared to controls and remained stable until day 7.
Based on this observation, other skin explants (3 donors) were pretreated with RW before exposing them to pollutants. After 24h (day 1), a highly concentrated mixture of pollutants was applied to the surface of the skin and culture was continued until day 2. The pollution mix was composed of 50 µg benzo(a)pyrene (B(a)P, 25 µg dibenzoanthracene (DBA), 10 µl benzene, 12,5 µl PM2.5 and 12,5 µl PM10. Titanium (Ti) and Lead (Pb) were selected as major components of PM2.5 and PM10 and represented 15 ppm and 12 ppm of the pollution mix respectively.
Skin viability and TEER were monitored on days 0, 1 and 2. Pollutants were quantified on the skin surface (wash), stratum corneum (SC), epidermis and dermis on day 2. Samples of the receptor fluid were collected 4, 6, 8, 10, 12 and 24 hours after exposure to pollution. High-performance liquid chromatography (HPLC) was used for B(a)P and DBA quantification, Gas Chromatography-Mass Spectrometry (GC-MS) for benzene and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) for Ti and Pb. Transcriptomic analysis using DNA array was performed on day 2.
Pollution led to a barrier disruption with a 14.6% decrease in TEER values observed on the pollution exposed explants compared to control. Pretreatment with RW fully protected the skin barrier showing no significant difference with the control skin. In addition, RW pretreatment significantly reduced the penetration of all pollutants. B(a)P and DBA were less detected in the SC and epidermis. Benzene, Ti and Pb were found significantly more concentrated at the skin surface (wash) and less in the skin layers. Interestingly, Ti and Pb could be detected in the receptor fluid but their penetration kinetic was slowed down by RW pretreatment. The pollution-induced cytotoxicity (-20% viability vs control) could be prevented by RW pretreatment. Transcriptomic analysis revealed that pollution activated the Aryl hydrocarbon receptor (AhR) pathway and xenobiotic metabolization signaling and increased the expression of genes related to cell death, apoptosis and necrosis. Compared to the pollution condition, RW pretreatment stimulated the expression of genes related to cell viability and survival pathways while those related to cell death, apoptosis and necrosis were decreased.
This model of pollutants penetration on a living skin in culture allowed to demonstrate that Réotier water can enhance the skin permeability barrier in just a few hours and limit pollutants penetration and their toxicity on skin.
Réotier water (RW) is a calcium-rich natural water from the French Alps, which has been used since ancient times to cure and soothe skin conditions. We previously showed on reconstructed human epidermis that RW could reinforce the permeability barrier by inducing tight junctions and cornified envelope proteins expression and limited the penetration of the Lucifer yellow (Cenizo V. IFSCC 2018).
To test whether RW was able to prevent the pollutants penetration, we setup a skin model of pollutants penetration. In a proprietary sterile device made of a hermetic cell, the skin explant was placed on a receptor compartment containing culture medium, allowing the culture for several days. We sprayed RW daily on the skin explants and measured the Trans-Epidermal Electrical Resistance (TEER) at different time points for 7 days. After only 5 hours, TEER had already increased by 30% (p<0.0001) in treated skin compared to controls and remained stable until day 7.
Based on this observation, other skin explants (3 donors) were pretreated with RW before exposing them to pollutants. After 24h (day 1), a highly concentrated mixture of pollutants was applied to the surface of the skin and culture was continued until day 2. The pollution mix was composed of 50 µg benzo(a)pyrene (B(a)P, 25 µg dibenzoanthracene (DBA), 10 µl benzene, 12,5 µl PM2.5 and 12,5 µl PM10. Titanium (Ti) and Lead (Pb) were selected as major components of PM2.5 and PM10 and represented 15 ppm and 12 ppm of the pollution mix respectively.
Skin viability and TEER were monitored on days 0, 1 and 2. Pollutants were quantified on the skin surface (wash), stratum corneum (SC), epidermis and dermis on day 2. Samples of the receptor fluid were collected 4, 6, 8, 10, 12 and 24 hours after exposure to pollution. High-performance liquid chromatography (HPLC) was used for B(a)P and DBA quantification, Gas Chromatography-Mass Spectrometry (GC-MS) for benzene and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) for Ti and Pb. Transcriptomic analysis using DNA array was performed on day 2.
Pollution led to a barrier disruption with a 14.6% decrease in TEER values observed on the pollution exposed explants compared to control. Pretreatment with RW fully protected the skin barrier showing no significant difference with the control skin. In addition, RW pretreatment significantly reduced the penetration of all pollutants. B(a)P and DBA were less detected in the SC and epidermis. Benzene, Ti and Pb were found significantly more concentrated at the skin surface (wash) and less in the skin layers. Interestingly, Ti and Pb could be detected in the receptor fluid but their penetration kinetic was slowed down by RW pretreatment. The pollution-induced cytotoxicity (-20% viability vs control) could be prevented by RW pretreatment. Transcriptomic analysis revealed that pollution activated the Aryl hydrocarbon receptor (AhR) pathway and xenobiotic metabolization signaling and increased the expression of genes related to cell death, apoptosis and necrosis. Compared to the pollution condition, RW pretreatment stimulated the expression of genes related to cell viability and survival pathways while those related to cell death, apoptosis and necrosis were decreased.
This model of pollutants penetration on a living skin in culture allowed to demonstrate that Réotier water can enhance the skin permeability barrier in just a few hours and limit pollutants penetration and their toxicity on skin.