Mitochondria-targeted phenolic antioxidants induce ROS-protective pathways in primary human skin fibroblasts

Phytochemical antioxidants like gallic and caffeic acid are constituents of the normal human diet that display beneficial health effects, potentially via activating stress response pathways. Using primary human skin fibroblasts (PHSFs) as a model, we here investigated whether such pathways were induced by novel mitochondria-targeted variants of gallic acid (AntiOxBEN₂) and caffeic acid (AntiOxCIN₄). Both molecules reduced cell viability with similar kinetics and potency (72 h incubation, IC50 ~23 μM). At a relatively high but non-toxic concentration (12.5 μM), AntiOxBEN₂ and AntiOxCIN₄ increased ROS levels (at 24 h), followed by a decline (at 72 h). Further analysis at the 72 h timepoint demonstrated that AntiOxBEN₂ and AntiOxCIN₄ did not alter mitochondrial membrane potential (Δψ), but increased cellular glutathione (GSH) levels, mitochondrial NAD(P)H autofluorescence, and mitochondrial superoxide dismutase 2 (SOD2) protein levels. In contrast, cytosolic SOD1 protein levels were not affected. AntiOxBEN₂ and AntiOxCIN₄ both stimulated the gene expression of Nuclear factor erythroid 2-related factor 2 (NRF2; a master regulator of the cellular antioxidant response toward oxidative stress). AntiOxBEN2 and ANtiOxCIN4 differentially affected the gene expression of the antioxidants Heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1). Both antioxidants did not protect from cell death induced by GSH depletion and AntiOxBEN₂ (but not AntiOxCIN₄) antagonized hydrogen peroxide-induced cell death. We conclude that AntiOxBEN₂ and AntiOxCIN₄ increase ROS levels, which stimulates NRF2 expression and, as a consequence, SOD2 and GSH levels. This highlights that AntiOxBEN₂ and AntiOxCIN₄ can act as prooxidants thereby activating endogenous ROS-protective pathways.