Dynamic monitoring of glutathione redox status in UV-B irradiated reconstituted epidermis: effect of antioxidant activity on skin homeostasis.

An "oxidative stress" should be considered as the consequence of a disbalance within the cutaneous tissue, between pro-oxidant processes and effective antioxidant defence systems and finally the result of a detrimental disturbance of the "cellular redox homeostasis". Instead of monitoring some particular impairment or a specific biological target of oxidative stress, it is preferable to examine the global effect of a stress, which is revealed by a loss of cellular homeostasis. The measurement of glutathione (relative amount of reduced (GSH), an oxidized form (GSSG)), offers a suitable approximation of the cellular redox status,since glutathione plays a pivotal role in the biological antioxidant systems. It is involved in the "first line " defence systems but also in repairing and recycling systems. This work aimed to develop an in vitro model that enables the dynamic monitoring of the glutathione redox status after UV-B irradiation. The method was performed in a commercially available Reconstituted Human Epidermis (RHE) model characterized by an high histo-morphological correspondence with normal human skin and by a very good inter batch reproducibility. With this experimental design it was possible to assess both the "physiological " behaviour of the epidermis after irradiation and also the ability of a tested ingredient to preserve or to restore the redox cutaneous homeostasis. The epidermis were irradiated and allowed to different recovery time (1,5,8,24 h) After each recovery time the quantification of GSH and GSSG by HPLC has been performed on RHE homogenates prepared in carefully controlled conditions. A series of non treated epidermis has been used as reference control. The redox homeostasis loss (e.g. GSH depletion) has been observed immediately after irradiation and remained between 1 and 5 h, although partial recovery takes place. In the presence of antioxidant actives the GSH depletion was minimised, and a faster restoration of homeostasis has been observed. The GSH/GSSG ratio was differentially modified by the actives tested, providing important insights into their specific mechanism of action. The glutathione redox status appears a very sensitive sensor, and kinetic studies have correlated protection against oxidative stress with redox homeostasis preservation.