IMPACT OF REAL-LIFE OZONE EXPOSURE ON SKIN IN VITRO AND IN VIVO
357
Presented by: Laurence DENAT
INTRODUCTION
Skin is one of the main organs directly exposed to environmental insults, especially pollution and ozone. At the ground level, Ozone (O3) is a gas produced by the action of ultraviolet radiations on primary pollutants mainly produced by human activities where its concentration can reach 0.1 ppm during peaks. Ozone is an extremely reactive molecule and constitutes the strongest oxidizing agent in contact with the skin in daily life. This pollutant directly interacts with lipids of epidermal surface layers and especially with unsaturated lipids present in sebum and stratum corneum (ozonolysis reaction), generating oxidized molecules, including very reactive aldehydes. Then follows a cascade of biochemical reactions and cellular responses impacting deeper layers of the skin like lipid peroxidation, endogenous antioxidant depletion, protein carbonylation, cellular stress and inflammation. Ozone exposure has been correlated with disruption of skin integrity and dermatological disorders like atopic dermatitis, but no causal link has been showed yet under real-life ozone exposure. To address this issue, the present study focused on evaluating the in vitro and in vivo impact of real-life concentrations of ozone on skin.
METHODS
Ozone generator consisted in an ultraviolet lamp generating photolysis of the oxygen molecule from air. Ozone was dispersed in a chamber inside a cell culture incubator or in a cylindrical cup fitted onto the skin of subjects.
In vitro: In vitro reconstructed human full thickness skin model (T-skinTM, Episkin, Lyon, France) was exposed to various concentrations of ozone (from 0.8 ppm to 0.1 ppm) either with 1, 2 or 3 exposures for 18 hours each. Reconstituted sebum (Synelvia, Toulouse, France; composition: free fatty acids, squalene, cholesterol, cholesterol esters, waxes, glycerides) was applicated on reconstructed skins at a concentration of 300µg/cm² before the first ozone exposure. Skin samples were analyzed for lipid oxidation by mass spectrometry, protein carbonylation by carbonyl score and protein expression by Western Blot.
In vivo: The upper back skin of 10 subjects was exposed to ozone, using specially designed cylindrical cups. Exposure protocol lasted 2 weeks and included 6 two-hour exposures to 0.3 ppm of ozone. Sebum and D-squame® samples were analyzed to determine lipid oxidation and protein expression by mass spectrometry, protein carbonylation by Elisa test analysis.
RESULTS
In order to study real-life ozone exposure, various ozone concentrations from 0.8 ppm to 0.1 ppm were used. Our results showed that ozone exposure induced lipid peroxidation and protein carbonylation. In addition, sebum application was exacerbating the impact of ozone on protein carbonylation. To go further with these mechanisms, biochemical markers of oxidation were analyzed. Ozone exposure induced an increase of different forms of oxidized lipids and amino acids. Moreover, after ozone exposure either with or without sebum, various proteins implicated in barrier function were found modulated and inflammation increased with higher levels of interleukins.
CONCLUSION
Ozone is a worldwide urban concern and it has been established that ozone concentration has been increasing since last years in many countries and will continue to increase with global climate warming. Maximal health threshold recommended by the World Health Organization is widely exceeded in a large part of the world. The current study brings for the first time new insights on impact of real-life ozone exposure conditions on skin. Some of these mechanisms could explain the increased number of patients with atopic dermatitis crisis observed after ozone peaks exposure. It is then key for consumers (i) to be aware of threats from ground-level ozone exposure for health but also for skin and (ii) to use daily topical application of specific cosmetical formulations that can protect cutaneous tissues against the impact of ozone exposure.
Skin is one of the main organs directly exposed to environmental insults, especially pollution and ozone. At the ground level, Ozone (O3) is a gas produced by the action of ultraviolet radiations on primary pollutants mainly produced by human activities where its concentration can reach 0.1 ppm during peaks. Ozone is an extremely reactive molecule and constitutes the strongest oxidizing agent in contact with the skin in daily life. This pollutant directly interacts with lipids of epidermal surface layers and especially with unsaturated lipids present in sebum and stratum corneum (ozonolysis reaction), generating oxidized molecules, including very reactive aldehydes. Then follows a cascade of biochemical reactions and cellular responses impacting deeper layers of the skin like lipid peroxidation, endogenous antioxidant depletion, protein carbonylation, cellular stress and inflammation. Ozone exposure has been correlated with disruption of skin integrity and dermatological disorders like atopic dermatitis, but no causal link has been showed yet under real-life ozone exposure. To address this issue, the present study focused on evaluating the in vitro and in vivo impact of real-life concentrations of ozone on skin.
METHODS
Ozone generator consisted in an ultraviolet lamp generating photolysis of the oxygen molecule from air. Ozone was dispersed in a chamber inside a cell culture incubator or in a cylindrical cup fitted onto the skin of subjects.
In vitro: In vitro reconstructed human full thickness skin model (T-skinTM, Episkin, Lyon, France) was exposed to various concentrations of ozone (from 0.8 ppm to 0.1 ppm) either with 1, 2 or 3 exposures for 18 hours each. Reconstituted sebum (Synelvia, Toulouse, France; composition: free fatty acids, squalene, cholesterol, cholesterol esters, waxes, glycerides) was applicated on reconstructed skins at a concentration of 300µg/cm² before the first ozone exposure. Skin samples were analyzed for lipid oxidation by mass spectrometry, protein carbonylation by carbonyl score and protein expression by Western Blot.
In vivo: The upper back skin of 10 subjects was exposed to ozone, using specially designed cylindrical cups. Exposure protocol lasted 2 weeks and included 6 two-hour exposures to 0.3 ppm of ozone. Sebum and D-squame® samples were analyzed to determine lipid oxidation and protein expression by mass spectrometry, protein carbonylation by Elisa test analysis.
RESULTS
In order to study real-life ozone exposure, various ozone concentrations from 0.8 ppm to 0.1 ppm were used. Our results showed that ozone exposure induced lipid peroxidation and protein carbonylation. In addition, sebum application was exacerbating the impact of ozone on protein carbonylation. To go further with these mechanisms, biochemical markers of oxidation were analyzed. Ozone exposure induced an increase of different forms of oxidized lipids and amino acids. Moreover, after ozone exposure either with or without sebum, various proteins implicated in barrier function were found modulated and inflammation increased with higher levels of interleukins.
CONCLUSION
Ozone is a worldwide urban concern and it has been established that ozone concentration has been increasing since last years in many countries and will continue to increase with global climate warming. Maximal health threshold recommended by the World Health Organization is widely exceeded in a large part of the world. The current study brings for the first time new insights on impact of real-life ozone exposure conditions on skin. Some of these mechanisms could explain the increased number of patients with atopic dermatitis crisis observed after ozone peaks exposure. It is then key for consumers (i) to be aware of threats from ground-level ozone exposure for health but also for skin and (ii) to use daily topical application of specific cosmetical formulations that can protect cutaneous tissues against the impact of ozone exposure.