Rotenone-exposure as cytofunctional aging model of human dermal fibroblast prior replicative senescence

• Published in: Toxicology in vitro Vol. 91; p. 105637
• Main Authors: da Cruz, Ivana Beatrice Mânica, de Afonso Bonotto, Nathália Cardoso, Turra, Bárbara Osmarin, Teixeira, Cibele Ferreira, Azzolin, Verônica Farina, Ribeiro, Ednea Aguiar Maia, Piccoli, Jacqueline Da Costa Escobar, Barbisan, Fernanda
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-09-2023
• Subjects: Aging; Cells, Cultured; Cellular Senescence; Collagen; Collagen metabolism; Fibroblasts; Humans; Oxidative metabolism; Rotenone - pharmacology; Skin aging; NO; Oxidative metabolism; DMSO; Pcarb; MTT; ATCC; OECD; fRA; Collagen metabolism; LPX; Ro; β-gal; Col-1; PBS; MN; qRT-PCR; DAPI; TBARS; Skin aging; SOD2; ECM; SpN; DCFH-DA; FGF-2; NDi; FGF-7; CPA; MMP-1; ROS; PI; HFF-1; NuD
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2023.105637

Rotenone (Ro), causes superoxide imbalance by inhibiting complex I of the mitochondrial electron transport chain, being able to serve as a model for functional skin aging by inducing cytofunctional changes in dermal fibroblasts prior to proliferative senescence. To test this hypothesis, we conducted an initial protocol to select a concentration of Ro (0.5, 1, 1.5, 2, 2.5, and 3 μM) that would induce the highest levels of the aging marker beta-galactosidase (β-gal) in human dermal HFF-1 fibroblasts after 72 h of culture, as well as a moderate increase in apoptosis and partial G1 arrestment. We evaluated whether the selected concentration (1 μM) differentially modulated oxidative and cytofunctional markers of fibroblasts. Ro 1.0 μM increased β-gal levels and apoptosis frequency, decreased the frequency of S/G2 cells, induced higher levels of oxidative markers, and presented a genotoxic effect. Fibroblasts exposed to Ro showed lower mitochondrial activity, extracellular collagen deposition, and fewer fibroblast cytoplasmic connections than controls. Ro triggered overexpression of the gene associated with aging (MMP-1), downregulation genes of collagen production (COL1A, FGF-2), and cellular growth/regeneration (FGF-7). The 1 μM concentration of Ro could serve as an experimental model for functional aging fibroblasts prior to replicative senescence. It could be used to identify causal aging mechanisms and strategies to delay skin aging events.
•Rotenone causes superoxide imbalance due mitochondrial dysfunction.•In fibroblasts superoxide imbalance induce oxidative stress, DNA damage.•Superoxide imbalance increases fibroblast β-gal activity.•Superoxide imbalance induces fibroblast extracellular matrix alterations.•Superoxide imbalance alters MMP1, FGF-2, FGF-7 and COL1A gene expression.•Alterations induced by rotenone have some similarities to aged-fibroblasts.