Generation of oxidative stress in human cutaneous models following in vitro ozone exposure

Ozone, one of the main components of photochemical smog, represents an important source of environmental oxidative stress. The skin, being the outermost barrier of the body, is directly exposed to environmental oxidant toxicants. Skin sebum and cellular plasma membrane lipids contain polyunsaturated fatty acids which are primary targets for ozone and free radical attack induced lipid peroxides. These ozonation processes in skin can also generate aldehydes, hydroxyhydroperoxides and specific Criegee's ozonides. In order to evaluate in vitro human skin susceptibility to ozone, we have exposed cultured immortalized human keratinocytes (DK7-NR) and the reconstructed human epidermis Episkin to 10 ppm of ozone in a specific incubator. We measured the formation of protein carbonyls by an ELISA method and monitored the oxidative stress using the fluorogenic probe 2',7'-dichlorofluorescin-diacetate (DCFH-DA). Results showed a time-dependent increase of fluorescence levels (linked to oxidative stress) in both models exposed to ozone. Using this protocol, we investigated the protective potential of different products including vitamin C, a thiol derivative and a plant extract. All products dramatically reduced oxidative responses during ozone exposure. Decreases observed in fluorescence levels were between 60 and 90% as compared to non-protected controls. These results demonstrate: (a) cutaneous in vitro models are remarkably susceptible to oxidative stress generated by an environmental air pollutant as ozone, and (b) raw antioxidants, thiols and vitamin C were efficient products to prevent ozone induced cellular oxidative damage.