Paradoxical enhancement of oxidative stress-induced DNA damage and impaired redox homeostasis by Glutaredoxin 1 overexpression in human HeLa cells

ABSTRACT Oxidants, including reactive oxygen species (ROS), oxidize intracellular molecules, such as lipids, proteins and deoxyribonucleic acid (DNA). Cysteine thiol groups in proteins are particularly susceptible to oxidation, forming intra- and inter-protein disulfides or disulfide bonds with glutathione (GSH). Glutaredoxin 1 (GRX1), an antioxidant enzyme, reduces these disulfide bonds by oxidizing and subsequently regenerating them via GSH, GSH reductase and reduced nicotinamide adenine dinucleotide phosphate (NADPH). While antioxidants protect cells from oxidative stress, excessive amounts may be harmful. This study investigated the effects of the overexpression of GRX1 in human tetracycline-regulated expression (T-REx) HeLa cells exposed to γ-irradiation, H2O2 or heat shock. GRX1-overexpressing cells exhibited reduced survival and impaired proliferation under these conditions relative to controls. Intracellular oxidant levels, measured using 2′, 7′-dichlorofluorescein diacetate, were elevated in these cells following oxidative stress. We conducted alkaline comet and micronucleus formation assays to assess DNA damage and measured γH2AX levels. The results revealed increased single-strand breaks and double-strand breaks in GRX1-overexpressing cells under oxidative stress. Additionally, in GRX1 overexpressing cells, NADPH recovery was delayed after stress, indicating impaired redox homeostasis. Moreover, the overexpression of GRX1 reduced the expression levels of the stress response factor JNK. This study highlights the complex role of GRX1 in cellular stress responses, suggesting that excessive antioxidant activity may paradoxically increase oxidative damage and impair stress adaptation.