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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

View larger version (19K): [in this window] [in a new window]   Figure 1. Enzyme-linked immunosorbent assay (ELISA) measurement showing the time-dependent secretion of tumour necrosis factor (TNF) into the culture supernatant. Endothelial cells were exposed to selected doses of low linear energy transfer (LET) radiation and harvested at the indicated time points. For the positive control, cells were incubated with 1 µg ml–1 lipopolysaccharide (LPS) and harvested at 16 h. Sandwich ELISA for TNF was carried out in 0.1 ml of culture supernatant. Cell counts were performed before harvesting, and TNF levels in picograms were normalized to 1 x 106 cells ml–1.

View larger version (42K): [in this window] [in a new window]   Figure 2. Survival of human aortic endothelial cells examined by either clonogenic assay or fold growth following: (a) a 5 h incubation with tumour necrosis factor (TNF) at the indicated doses or (b) exposure to 0.1 Gy, 1 Gy and 2 Gy. Clonogenic assay was performed as described in Materials and methods. Cell viability was determined by the trypan blue dye exclusion method. The data presented are the mean ± standard error of the mean (SEM) of at least four independent experiments. (c) Morphology of endothelial cells. Cells were either untreated or treated with selected concentrations of TNF and stained with acridine orange and ethidium bromide showing the absence of apoptotic cells. Cells incubated with 10 µM staurosporine (positive control) show visible apoptotic cells.

View larger version (93K): [in this window] [in a new window]   Figure 3. (a) Intracellular oxidative stress examined by immunofluorescence. Endothelial cells preincubated with 2',7'-dichlorofluorescein diacetate (DCFH-DA; 10 µM) for 30 min were either mock treated, treated with recombinant tumour necrosis factor (TNF) or 100 µM H2O2 alone or in combination with polyethylene glycol-tagged superoxide dismutase (PEG-SOD) and polyethylene glycol-tagged catalase (PEG-CAT). The cells were examined 30 min after treatment at 200x magnification under a green channel (at excitation 488 nm and emission 570 nm) using a Nikon TE-2000U epifluorescence microscope. Brightfield images, showing the total number of cells present in the same field, were obtained under phase contrast at 200x magnification. (b) Histogram showing the levels of TNF-induced intracellular reactive oxygen species (ROS). Cells preincubated with DCFH-DA (10 µM) for 30 min were (i) mock treated, (ii) treated with 0.1 ng ml–1, 1 ng ml–1 and 10 ng ml–1 TNF, (iii) treated with 100 µM H2O2 or (iv) exposed to 2 Gy. The fluorescent intensity was measured 30 min after treatment or exposure by flow cytometry. The percentage of cells positive for fluorescein was calculated from the total number of cells gated. The data presented are means ± standard deviation (SD) of three independent experiments. The p-values were calculated using Student's t-test.

View larger version (22K): [in this window] [in a new window]   Figure 4. Chromosome- and chromatid-type aberrations measured by solid-stained chromosome analysis at day 3, 12 and 20 post exposure to either tumour necrosis factor (TNF) or X-rays at the indicated doses. Data are presented as the mean aberrations per cell. Day 3 data were pooled from five separate experiments, while days 12 and 20 were pooled from three separate experiments. The asterisks denote the p-value 0.05 compared with corresponding negative controls by chi-squared analysis for day 3 and Fisher's exact test for days 12 and 20.

View larger version (19K): [in this window] [in a new window]   Figure 5. Chromosome damage in human aortic endothelial cells as measured by the alkaline comet assay after exposure to radiation or after treatment with tumour necrosis factor(TNF). Cells exposed to 2 Gy or treated with 100 µM H2O2 were used as positive controls. Cells were treated with TNF at the indicated concentrations and trypsinized after 5 h for the comet assay. Nucleic acid was stained with SYBR Gold, and Komet 5.5 image analysis software was used to determine the percentage DNA in the comet tails of each cell examined. The data are presented as the average percentage tail DNA pooled from four separate experiments, and the p-values were determined by a two-sample t-test. Error bars represent the standard error of the mean (SEM). (a) TNF-exposed cells only with the indicated positive controls. (b) Cells were either treated as before or pretreated with 1000 units ml–1 catalase for 1 h prior to the addition of TNF or exposure to X-rays or H2O2. (c) Cells were either treated as before or pretreated with both 150 µM pyrrolidine dithiocarbamate (PDTC) and 20 mM N-acetyl-L-cysteine (NAC) for 1 h prior to the addition of TNF or exposure to X-rays or H2O2.

View larger version (14K): [in this window] [in a new window]   Figure 6. The possible mechanism of how radiation-induced tumour necrosis factor (TNF) could cause genomic instability in the non-targeted cells as a bystander response.