anhydrovitamin-a and retinol-palmitate

We have previously reported that photoirradiation of retinyl palmitate (RP) in ethanol with UVA light results in the formation of photodecomposition products, including 5,6-epoxy-RP and anhydroretinol (AR). Photoirradiation in the presence of a lipid, methyl linoleate, induced lipid peroxidation, suggesting that reactive oxygen species (ROS) are formed. In the present study, we employ an electron spin resonance (ESR) spin trap technique to provide direct evidence as to whether or not photoirradiation of RP by UVA light produces ROS. Photoirradiation of RP by UVA in the presence of 2,2,6,6-tetramethylpiperidine (TEMP), a specific probe for singlet oxygen, resulted in the formation of TEMPO, indicating that singlet oxygen was generated. Both 5,5-dimethyl N-oxide pyrroline (DMPO) and 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) are specific probes for superoxide. When photoirradiation of RP was conducted in the presence of the DMPO or BMPO, ESR signals for DMPO-*OOH or BMPO-*OOH were obtained. These results unambiguously confirmed the formation of superoxide radical anion. Consistent with a free radical mechanism, there was a near complete and time-dependent photodecomposition of RP and its photodecomposition products. ESR studies on the photoirradiation of 5,6-epoxy-RP and AR indicate that these compounds exhibit similar photosensitizing activities as RP under UVA light.

Topics: Antioxidants; Diterpenes; Electron Spin Resonance Spectroscopy; Linoleic Acids; Lipid Peroxidation; Retinyl Esters; Singlet Oxygen; Superoxides; Ultraviolet Rays; Vitamin A

Retinyl palmitate (RP) is an ester of retinol (vitamin A) and the predominant form of retinol found endogenously in the skin. We have previously reported that photoirradiation of RP with UVA light resulted in the formation of anhydroretinol (AR), 5,6-epoxyretinyl palmitate (5,6-epoxy-RP) and other photodecomposition products. While AR was formed through an ionic photodissociation mechanism, 5,6-epoxy-RP was formed through a light-mediated, free radical-initiated chain reaction. In the current study, the phototoxicity of RP, AR and 5,6-epoxy-RP in human skin Jurkat T-cells with and without light irradiation was determined using a fluorescein diacetate assay. Under similar conditions, the Comet assay was used to assess damage to cellular DNA. Nuclear DNA was not significantly damaged when the cells were irradiated by UVA plus visible light in the absence of a retinoid; however, when the cells were illuminated with UVA plus visible light in the presence of either RP, 5,6-epoxy-RP or AR (50, 100, 150 and 200 microM), DNA fragmentation was observed. Cell death was observed for retinoid concentrations of 100 microM or higher. When treated with 150 microM of RP, 5,6-epoxy-RP or AR, cell death was 52, 33 and 52%, respectively. These results suggest that RP and its two photodecomposition products, AR and 5,6-epoxy-RP, induce DNA damage and cytotoxicity when irradiated with UVA plus visible light. We also determined that photoirradiation of RP, AR and 5,6-epoxy-RP causes single strand breaks in supercoiled phi chi 174 plasmid DNA. Using a constant dose of UVA light (50 J/cm2), the level of DNA cleavage was highest in the presence of AR, followed by 5,6-epoxy-RP, then RP. The induced DNA strand cleavage was inhibited by NaN3. These results suggest that photoirradiation of RP, 5,6-epoxy-RP and AR with UVA light generates free radicals that initiate DNA strand cleavage.

Topics: Anticarcinogenic Agents; Cell Survival; Comet Assay; Dermatitis, Phototoxic; Diterpenes; DNA Damage; Humans; Retinyl Esters; Skin; Sunlight; Vitamin A

Topics: Chromatography, High Pressure Liquid; Diterpenes; Humans; Liver; Reference Standards; Retinyl Esters; Spectrophotometry, Ultraviolet; Vitamin A