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Oxidative stress signalling: a potential mediator of tumour necrosis factor -induced genomic instability in primary vascular endothelial cells

Departments of ¹ Radiation Oncology, ² Otolaryngology Head & Neck Surgery and ⁴ Pathology, University of Texas Health Science Center, San Antonio, TX, ³ Environmental Toxicology Graduate Program, University of California, Riverside, CA, USA and ⁵ Radiation and Genome Stability Unit, Medical Research Council, Harwell, Oxford, UK

Correspondence: Mohan Natarajan, Associate Professor, Department of Radiation Oncology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. E-mail: Natarajan{at}uthscsa.edu

Studying the potential role of tumour necrosis factor (TNF) in the initiation of genomic instability is necessary to understand whether TNF can serve as a signalling mediator of radiation-induced genomic instability in non-irradiated bystander cells. In this study, we examined whether TNF could initiate processes through oxidative stress signalling that lead to DNA damage and genomic instability in primary vascular endothelium. In these cells, low linear energy transfer (LET) radiation (0.1–2 Gy) induced the secretion of TNF into the culture medium. When added ectopically, TNF at concentrations ranging from 0.1 ng ml^–1 to 10 ng ml^–1 increased (twofold to threefold) intracellular oxidative stress. Next, to examine whether TNF induces genetic damage, cells were treated with TNF for 5 h and analysed immediately using the single cell gel electrophoresis assay or after 3 days, 12 days and 20 days using solid stain chromosomal analysis. Cells exposed to 0.1 Gy, 1 Gy or 2 Gy or treated with 100 µM H₂O₂ were used as positive controls. The results showed that TNF as low as 0.1 ng ml^–1 could initiate increased DNA damage compared with untreated controls. When examined in the progeny cells after several generations, the chromosomal instability appeared to be carried over even after day 12 and day 20. The increased genetic damage is inhibited in cells that are pre-incubated with the antioxidant enzyme catalase, the antioxidant N-acetyl-L-cysteine or the metal chelator pyrrolidine dithiocarbamate. These results clearly indicate that TNF at concentrations at which no cytotoxicity is observed could induce genetic damage through free radical generation, which could, in turn, lead to the delayed events associated with genomic instability.