Advancing EPR spectroscopy with BMPO for UVA-induced radical detection in skin: Refining spin trapping and uncovering glutathione-dependent oxidative mechanisms

Ultraviolet A (UVA) irradiation significantly impacts skin health by generating free radicals, including reactive oxygen species (ROS), lipid oxygen species (LOS), and carbon-centered radicals (CCR), contributing to oxidative stress. Electron paramagnetic resonance (EPR) spectroscopy enables the direct detection of these radicals, using spin traps like 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO). While DMPO is suitable for detecting short-lived species such as hydroxy (•OH) and alkoxy radicals, BMPO offers greater stability, particularly for superoxide (O₂^-) and hydroperoxyl radicals (•OOH). This study refines EPR-based protocols for radical detection in UVA-irradiated skin by comparing DMPO and BMPO, revealing a shift from short-lived ROS to more stable LOS with increasing UVA-exposure. For the first time, the glutathione (GSH)-mediated conversion of O₂^- to •OH was directly quantified via spin trapping in skin tissue. Although GSH functions as a central antioxidant in skin, it indirectly promotes •OH formation via the Fenton reaction under UV-induced oxidative stress, potentially contributing to tissue damage. BMPO's enhanced stability as a spin trap for O₂^- in skin tissue enables precise detection of this GSH-dependent radical transformation, offering new insights into protective and damaging mechanisms under oxidative conditions. A standardized protocol for ex situ UVA irradiation of skin and subsequent radical measurement was developed, establishing a foundation for future studies with other stress factors. This research refines spin trapping methodologies and advances the understanding of UVA-induced oxidative processes, offering a framework for future dermatological and photobiological investigations.