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ROS‐challenged keratinocytes as a new model for oxidative stress‐mediated skin diseases
Author(s) -
Barygina Victoria,
Becatti Matteo,
Lotti Torello,
Moretti Silvia,
Taddei Niccolò,
Fiorillo Claudia
Publication year - 2019
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.27485
Subject(s) - hacat , reactive oxygen species , nadph oxidase , superoxide , oxidative stress , catalase , superoxide dismutase , extracellular , keratinocyte , intracellular , chemistry , hydrogen peroxide , biochemistry , microbiology and biotechnology , nitric oxide , in vitro , biology , enzyme , organic chemistry
Abstract In the current study, the effects of the reactive oxygen species (ROS) generator 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH) on extracellular and intracellular ROS production in human keratinocytes (HACAT) were studied. AAPH is a water‐soluble compound able to generate ROS at known and constant rates at 37°C. The short treatment (2 h) with AAPH brought a significant dose‐dependent increase in NADPH oxidase activity in intact keratinocytes. The long‐term treatment (24 h) with AAPH led to a persistent increase in NADPH oxidase activity for up to 48 hour following the AAPH removal from cell incubation medium. ROS and nitric oxide levels, lipoperoxidation, intracellular calcium, mitochondrial superoxide production, and membrane potential were significantly modified in AAPH‐treated HACAT. Superoxide dismutase (SOD) and/or catalase addition to HACAT revealed that untreated keratinocytes produce mostly superoxide anion (O 2 − ), while AAPH‐treated keratinocytes overproduce hydrogen peroxide (H 2 O 2 ) in extracellular medium. H 2 O 2 is particularly stable and plays important roles in several cell signaling pathways. Taken together, our findings suggest a cost‐effective and easily reproducible in vitro model of stressed human keratinocytes releasing significantly elevated ROS amounts in extracellular medium with respect to control keratinocytes. The possible application of the proposed model for keratinocytes‐melanocytes cross‐talk studies is also suggested. The model of AAPH‐stressed human keratinocytes described here can represent a useful tool for redox cross‐talk studies between keratinocytes and other skin cell types, and applied for researches regarding skin pathologies associated with oxidative stress.