Research Paper Volume 12, Issue 2 pp 1910—1927
Effect of antioxidants on the H2O2-induced premature senescence of human fibroblasts
- 1 Department of Analytical Biochemistry, Institute of Food Technology and Nutrition, College of Natural Sciences, Rzeszow University, Rzeszow, Poland
- 2 Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland
- 3 Cytometry Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection University of Lodz, Lodz, Poland
Received: October 29, 2019 Accepted: January 2, 2020 Published: January 21, 2020
https://doi.org/10.18632/aging.102730How to Cite
Abstract
The study was aimed at evaluation of the role of secondary oxidative stress in the stress-induced premature senescence (SIPS) of human fibroblasts induced by H2O2. Two fibroblast lines were used: lung MRC-5 and ear H8F2p25LM fibroblasts. The lines differed considerably in sensitivity to H2O2 (IC50 of 528 and 33.5 μM, respectively). The cells were exposed to H2O2 concentrations corresponding to IC50 and after 24 h supplemented with a range of antioxidants. Most of antioxidants studied slightly augmented the survival of fibroblasts at single concentrations or in a narrow concentration range, but the results were not consistent among the cell lines. Chosen antioxidants (4-amino-TEMPO, curcumin, caffeic acid and p-coumaric acid) did not restore the level of glutathione decreased by H2O2. Hydrogen peroxide treatment did not induce secondary production of H2O2 and even decreased it, decreased mitochondrial potential in both cell lines and induced changes in the mitochondrial mass inconsistent between the lines. Antioxidant protected mitochondrial potential only in H8F2p25LM cells, but attenuated changes in mitochondrial mass. These results speak against the intermediacy of secondary oxidative stress in the SIPS induced by H2O2 and suggest that the small protective action of antioxidants is due to their effects on mitochondria.
Abbreviations
4-amino-TEMPO: 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl; DMEM: Dulbecco’s Modified Eagle Medium; DMEM+GlutaMax: Dulbecco’s Modified Eagle Medium + GlutaMax; DMSO: dimethyl sulfoxide; GSH: L-glutathione reduced; H2DCF-DA: 2,7’-dichlorodihydro-fluorescein diacetate; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; NAO: Acridine Orange 10-nonyl bromide; NEM: N-ethylmaleimide; OPA: ortho-phthal-dialdehyde; PBS: phosphate-buffered saline; ROS: reactive oxygen species; SIPS: stress-induced premature senescence; TCA: trichloroacetic acid; TEMPO: 2,2,6,6-tetramethyl-piperidine-1-oxyl; TEMPOL: 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; Δψm:: mitochondrial membrane potential; IC50: half maximal inhibitory concentration: undefined.