j. Cosmet. Sci., 54, 589-598 (November/December 2003) Bioconvertible vitamin antioxidants improve sunscreen photoprotection against UV-induced reactive oxygen species KERRY M. HANSON and ROBERT M. CLEGG, Laboratory for Fluorescence Dynamics, Department of Physics, University of Illinois, Urbana-Champaign, Illinois. Accepted for publication May 20, 2003. Synopsis The ability of sunscreens and antioxidants to deactivate highly destructive reactive oxygen species in human skin has remained inconclusive. Two-photon fluorescence imaging microscopy was used to determine the effect of sunscreen/antioxidant combinations upon UV-induced ROS generation in ex vivo human skin. A sunscreen combination containing octylmethoxycinnamate (Parsol © MCX) and avobenzone (Parsol © 1789) at SPF 8 and SPF 15 was tested for its ability to prevent UV radiation from generating ROS in the viable epidermal strata ofex vivo human skin. A UV dose equivalent to two hours of North American solar UV was used to irradiate the skin. Each sunscreen reduced the amount of ROS induced in the viable strata by a value consistent with the SPF level. UV photons that were not absorbed/scattered by the sunscreen formulations generated ROS within the viable epidermal layers. The addition of the bioconvertible antioxidants vitamin E acetate and sodium ascorbyl phosphate (STAY-C © 50) improves photoprotection by converting to vitamins E and C, respectively, within the skin. The bioconversion forms an antioxidant reservoir that deactivates the ROS generated (within the strata granu- losum, spinosum, and basale) by the UV photons that the sunscreens do not block in the stratum corneum. INTRODUCTION Ultraviolet (UV) irradiation of human skin induces the generation of reactive oxygen species (ROS), including singlet oxygen (102) , hydrogen peroxide (H202) , and/or per- oxynitrite (ONOO-) (1). These highly reactive derivatives of molecular oxygen react with cellular components including lipid membranes and are considered a source of photoaging and skin cancers that appear later in life (2-9). The ability of sunscreens to protect against the generation of ROS within the skin has not been identified. Although sunscreens do prevent erythema, and are recommended to be used as part of safe-sun practices (10), current research suggests that photoprotection is also needed to reduce ROS levels within the skin (11,12). Address all correspondence to Kerry M. Hanson. 589
590 JOURNAL OF COSMETIC SCIENCE Antioxidants have recently been added to formulations to deactivate ROS levels (12) however, similar to our limited understanding of sunscreen-mediated ROS photopro- tection, whether or not the addition of antioxidants enhances photoprotection against UV-induced ROS generation is not well understood. This can be attributed predomi- nantly to limitations in technology that, until recently, have not allowed for the study of cellular processes within the opaque and heterogeneous environment of the skin. Clearly, understanding the effect of sunscreens and antioxidants upon the level of UV- induced ROS generated under biologically relevant conditions (i.e., in viable human skin, at a commonly obtained UV dose) will aid our understanding of the efficacy of these formulations and lead to improved photoprotection. We have recently developed a two-photon fluorescence microscopy method with sub- micron spatial resolution and submillimeter depth penetration in live human ex viva skin to determine UV-induced ROS levels (1). Following UV irradiation, at a dose equivalent to two hours of North American summer solar UV (1600 J m-2), of human breast tissue, ca. 10 -4 moles of •O 2, H202 and/or ONOO- were generated within each epidermal stratum (1). As discussed herein, two-photon fluorescence microscopy serves as an excellent tool to determine the photoprotective effects of sunscreens and antioxidants upon ROS levels generated in epidermis irradiated by a commonly obtained UV dose (1600 J m-2). MATERIALS AND METHODS MATERIALS Reagents. N,N' -[[3 ',6' -bis(acetyloxy)- 3-oxospiro[isobenzofuran- 1 (3 H),9' -[9H]xanthene]- 2••7•-diy•]bis(methy•ene)].bis•N.[2.[(acety••xy)meth•xy]-2.•x•ethy•]].bis[(acety••xy)- methyl] (calcein-AM), dihydrorhodamine-123 (DHR) and fluorescein diphosphate (FDP) were obtained from Molecular Probes. Sunscreen/antioxidant combinations were provided by Roche Vitamins Inc. Table I lists the active ingredients studied. Two sunscreen formulations with different sun protection factors (SPF) were tested. All ingredients are listed by %-weight. The SPF 8 sunscreen contained 4% octylmethoxy- cinnamate (OMC, Parsol © MCX) and 2% avobenzone (Parsol © 1789). The SPF 15 sunscreen contained 7.5% OMC and 3% Parsol 1789. Vitamin E acetate and sodium ascorbyl phosphate were added to the formulations at 2.5 %. Skin samples. Ex viva human skin (breast and facial) was obtained following patient- requested surgery. Samples were obtained with approval from the University of Illinois Internal Review Board. Skin from two individuals was studied. Both skin samples were Table I Active Ingredients Listed by %-Weight SPF 8 SPF 15 Ingredient -E/C +E +E/C -E/C +E +E/C Octylmethoxycinnamate (Parsol © MCX) Avobenzone (Parsol © 1789) Vitamin E acetate Sodium ascorbyl phosphate (STAY-C © 50) 4 4 4 7.5 7.5 7.5 2 2 2 3 3 3 0 2.5 2.5 0 2.5 2.5 0 0 2.5 0 0 2.5
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