Topically applied vitamin C and cysteine derivatives protect against UVA-induced photodegradation of suprofen in ex vivo pigskin.

Exposure of the nonsteroidal anti-inflammatory drug suprofen (SUP) to UV-radiation results in the formation of radicals, reactive oxygen species (ROS), photodecarboxylated products and photoadducts with biomacromolecules. Using an ex vivo pigskin explant model, we investigated whether topical coapplication of the water-soluble antioxidants vitamin C (Lascorbic acid, ASC), N-acetyl-L-cysteine (NAC) or L-cysteine ethylester (CYSET) with SUP reduced ultraviolet A (UVA)-induced decomposition of SUP. UVA-induced changes in antioxidant bioavailability in the stratum corneum and epidermis were also studied. Epidermal bioavailability of SUP in sham-irradiated pigskin increased 2.2- to 4.1-fold after the lowest antioxidant doses (P < 0.05). As compared with no applied antioxidant, increasing doses of all tested antioxidants resulted in increased levels of SUP and decreased levels of photoproducts (P < 0.05). A maximal protection against SUP photodegradation of 70% was found after an ASC dose of 1 micromol/cm2; these values were 60% for a NAC dose of 10 micromol/cm2 and 50% for a CYSET dose of 5 micromol/cm2. Skin antioxidant levels increased with increasing applied dose (P < 0.05); the bioavailability of CYSET was approximately three-fold lower than that of ASC and NAC. UVA exposure resulted in 30-50% consumption of the topically applied ASC or NAC in the stratum corneum, whereas CYSET was not consumed. In conclusion, the topically applied water-soluble antioxidants ASC, NAC and CYSET protect against UVA-induced decomposition of SUP by scavenging radicals and ROS. Coapplication of these antioxidants may therefore be an effective way to reduce or prevent the phototoxic effects of SUP in vivo.

Photoprotection by antioxidants against UVB-radiation-induced damage in pig skin organ culture.

Topically applied antioxidants constitute an important group of protective agents against skin damage induced by ultraviolet radiation. The current study was performed to investigate whether a recently developed ex vivo pig skin model was suitable for short-term studies of the mechanism(s) of UVB-radiation-induced skin damage; the protective effect of topical application of alpha-tocopherol, l-ascorbic acid, alpha-lipoic acid, glutathione ethylester and N-acetylcysteine was tested. Increasing doses of the antioxidants were applied topically on ex vivo pig skin explants and allowed to penetrate for 60 min. Epidermal antioxidant bioavailability was measured before and 60 min after exposure to an ultraviolet B (UVB) radiation of 7.5 kJ/m2. Cell viability (trypan blue dye exclusion) and apoptosis were measured 48 h later in isolated keratinocytes. UVB-radiation-induced epidermal lipid peroxidation was determined immediately after exposure of the skin to a UVB dose of 28 kJ/m2. All antioxidants tested became bioavailable in pig skin epidermis, and none of them were depleted after UVB-radiation exposure. Increasing doses of the antioxidants tested decreased UVB-radiation-induced cell death and apoptosis. The highest doses of antioxidants prevented UVB-radiation-induced lipid peroxidation; alpha-lipoic acid only tended to decrease lipid peroxidation. In conclusion, a single topical dose of the above antioxidants on ex vivo pig skin can reduce UVB-radiation-induced oxidative stress and lipid peroxidation and thereby reduce apoptotic stimuli and cell death. Furthermore, the ex vivo pig skin model was a useful tool for testing compounds for their antioxidant activity.

Apoptosis in leukocytes induced by UVA in the presence of 8-methoxypsoralen, chlorpromazine or 4,6,4'-trimethylangelicin.

Although 8-methoxypsoralen (8-MOP) has been successfully used in extracorporeal photochemotherapy (ECP) of several T cell-mediated diseases, the exact mechanism of the drug therapeutic action has not been established. We have studied in vitro apoptotic activity of 8-MOP, and for comparison of 4,6,4'-trimethylangelicin (TMA) and chlorpromazine (CPZ) as alternative photosensitizers for potential use in photopheresis. However, while 8-MOP and CPZ are known for their immune suppressive activity, TMA does not exhibit such an activity in an animal model for ECP. Apoptosis and necrosis were measured in both Jurkat cells and primary rat leukocytes under conditions comparable to those used in the animal model to suppress contact hypersensitivity (CHS). Cells were irradiated with UVA (200 kJ/m(2)) after treatment with 8-MOP, CPZ or TMA (300 ng/ml). Flow cytometric analysis (annexin-V-FLUOS/propidium iodide) and fluorescence microscopy examinations, using acridine orange/propidium iodide, indicated that the number of cells undergoing apoptosis or necrosis increased significantly after 24 h following treatment. Similar results were observed irrespective of the cell type and photosensitizer used. The results of the present study, combined with previous observations with the animal model for ECP, suggest that apoptosis is not likely to be a critical step in the cascade of events leading to immunosuppression.