Exposure of normal human epidermal keratinocytes to ozone results in increased expression of cytochrome P450 through activation of aryl hydrocarbon receptor

595 Ozone, one of the main components of photochemical smog, represents an important source of environmental oxidative stress to which the skin is exposed, especially during smoggy and ozone alert days. The exposure of ozone has been shown to induce antioxidant depletion as well as the oxidation of lipids and proteins in mouse skin. The primary role of cytochrome P450s (CYPs) in cutaneous tissue is to participate in biotransformation of endogenous substrates and xenobiotic materials such as environmental pollutants to which human skin is either deliberately or inadvertently exposed. For example, induction of the CYP1 family of isoforms in the skin by environmental pollutants and ultraviolet radiation has been suggested to be responsible for enhanced cutaneous carcinogenesis. The present study was designed to determine the effect of ozone on CYP isoforms, aryl hydrocarbon receptor (AhR), epidermal growth factor receptor (EGFR) and its downstream events in normal human epidermal keratinocytes (NHEK). Western blotting and RT-PCR analyses indicated that ozone (0.3 ppm for 20 min) exposure to NHEK resulted in increased protein and mRNA expression of CYP1A1, CYP1A2 and CYP1B1, the CYP isoforms responsible for metabolic activation of environmental pollutant such as polycyclic aromatic hydrocarbons present in cigarette smoke. Because AhR induces transcription of CYP1A genes, we determined the effect of ozone on AhR. Ozone exposure resulted in increased expression of AhR protein and mRNA in NHEK. Since AhR is known to “cross-talk” with the EGFR, we next examined the phosphorylation of EGFR in ozone exposed cells. Exposure of NHEK to ozone resulted in an increased phosphorylation of EGFR. Next we determined the effect of ozone on downstream events of EGFR and found that it resulted in an increased (i) activation of PI3K, (ii) phosphorylation of AKT, and (iii) phosphorylation of ERK1/2, JNK1/2 and p38. To determine whether ozone-induced CYPs expression is EGFR and/or AhR-dependent, we silenced NHEK by appropriate siRNAs for EGFR and AhR and then exposed them to ozone. We found that AhR silencing by siRNA abolished the capacity of these cells to increase the protein and mRNA expressions of CYP1A1, CYP1A2 and CYP1B1 upon ozone exposure. However, silencing of EGFR did not show any effect on ozone-induced protein and mRNA expression of CYPs. These data indicate that induction of CYPs by ozone is mediated through activation of AhR and not by EGFR. These studies could lead to development of novel skin care products for ameliorating ozone-induced toxic effects in the skin. Based on these results, we suggest that supplementation of skin care products with inhibitors of CYPs might be beneficial to skin damages caused by ozone especially, during smoggy and ozone alert days.