Exposure of the skin to air pollutants is associated with skin aging and inflammatory or allergic skin conditions. Air pollution induces oxidative stress and collagen matrix degradation. Visible signs of aging and inflammation are a major concern for many people. Responding to people expectations requires identification and characterization of the main biological mechanisms associated with air pollution exposure, and establishment of standardized and flexible in vitro assays investigating the protective effects of skin care products. We introduce here different in vitro assays on fibroblasts and 3D skin models exposed to standardized urban dust and cigarette smoke extracts.
2. Methods and Results
We present functional assays on fibroblasts and 3D models exposed to standardized urban dust (UBD) and cigarette smoke extracts (CSE). The concentrations of these reference pollutants have been selected based on their biological relevance and do not induce cytotoxicity as measured by tetrazolium salts reduction assays. The effects of the pollutants on the chosen biomarkers were compared with reference compounds known for their protective effects, including carnosic acid and sulforaphane.
We used skin explants and 3D skin models to assess skin barrier function and hydratation levels of the skin under exposure to our reference pollutants.
The assays on dermal fibroblasts showed effects of air pollutants on skin ageing biomarkers including modulation of the expression levels of matrix metalloproteinase 1 and collagen 1. Using different models including primary dermal fibroblasts and epidermal keratinocytes as well as3D models, we addressed the effects of pollutants on the oxidative stress level using an oxidant-sensitive fluorescent probe. We also validated a panel of biomarkers involved in eicosanoids metabolism, inflammation, as well as detoxification and antioxidant pathways.
The assays are analyzed through fluorimetry, ELISA, quantitative PCR, and immunohistochemistry.
The data demonstrated that low concentrations of pollutants present no oxidative stress per se, yet increased drastically the oxidative stress levels of cells exposed to UVA (10J/cm²). This oxidative stress is inhibited in presence of vitamin C as well as carnosic acid or sulforaphane.
After 72h of exposure, the pollutants also decreased expression of biomarkers of skin firmness and strength such as elastin and collagen 1. Pollutants under study also increased expression of inflammation inducers such as cyclooxygenase-2 and 5-lipoxygenase, and induced expression of enzymes involved in extracellular matrix degradation.
Treatments with protective molecules against pollution effects induced expression of antioxidant enzymes HMOX1 and NQO1. The induction of MMP1 expression was also inhibited in presence of carnosic acid.
3. Discussion and Conclusion
Our data shed light on a better characterization of the effects of air pollutants on skin and demonstrated the potential of in vitro assays to assess the anti-pollution and detoxification potential of investigational compounds. The assays are currently available on normal human dermal fibroblasts, epidermal keratinocytes, and 3D skin models. We are currently optimizing assays to assess the effects of anti-pollution compounds on the air pollution-induced carbonylation, skin hydratation and epidermal water loss as well as the integrity of the skin barrier and associated biomarkers.