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British Journal of Radiology (2007) 80, S13-S22
© 2007 British Institute of Radiology
doi: 10.1259/bjr/15316848
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Full paper

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

M Natarajan, PhD 1,2 C F Gibbons, MS 3 S Mohan, PhD 4 S Moore, PhD 5 and M A Kadhim, PhD 5

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


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Studying the potential role of tumour necrosis factor (TNF){alpha} in the initiation of genomic instability is necessary to understand whether TNF{alpha} can serve as a signalling mediator of radiation-induced genomic instability in non-irradiated bystander cells. In this study, we examined whether TNF{alpha} 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{alpha} into the culture medium. When added ectopically, TNF{alpha} 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{alpha} induces genetic damage, cells were treated with TNF{alpha} 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{alpha} 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{alpha} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Cells initially surviving irradiation that are capable of proliferation may produce descendants with de novo chromosome aberrations and gene mutations. The accumulation of new genetic damage in successive progeny that leads to genetic instability may predispose the cells to carcinogenic processes. We have demonstrated previously the occurrence of genomic instability after exposure to radiation of various qualities [1–4]. Recently, our group and others have shown the induction of genomic instability in the progeny of cells that have never been irradiated but co-cultured with irradiated cells [4, 5]. Chromosomal instability was expressed in the progeny of more clonogenic haemopoietic stem cells than were traversed by an {alpha}-particle [4]. This was later confirmed using a grid to shield non-irradiated cells from irradiated cells [5]. These observations are collectively referred to as the "bystander effect". The significance of radiation-induced bystander effects, although not recognized initially, has gained importance in recent years. Earlier studies primarily obtained evidence by exposing cells in culture to low fluence broad beam irradiation. Reliance on probability estimates from data provided by conventional broad beam irradiators was recently superseded by demonstrating direct evidence of the occurrence of the "bystander effect" with the use of a single-particle/single-cell microbeam [6–9]. The microbeam allows one to define precisely which cell nuclei and what proportion of cell nuclei are traversed by an exactly defined number of {alpha}-particles [6]. Through these studies, the bystander response is thought to be dependent on cellular communication processes. The communication has been shown both in systems where irradiated cells are in contact with one another through gap junction phenomena [10, 11] as well as through soluble factors when cells are at considerable distances apart from each other [9, 12–14].

In reviewing the literature, it is speculated that gap junction communication might play a major role in the bystander response when cells are in close contact whereas, when the cells are not in contact, signalling processes mediated through soluble factors in the medium may play a predominant role [15]. Several soluble factors have been considered as potential candidates in the bystander response [9, 16, 17]. However, very little is known about the nature of the signalling mediators and their targets in non-irradiated cells. In this study, we examined whether a proinflammatory cytokine, tumour necrosis factor-alpha (TNF{alpha}), when added ectopically, could serve as one such mediator that could communicate with the intracellular target(s) and cause genomic instability. It is widely known that radiation can trigger TNF{alpha} expression in many cell types. TNF{alpha} signalling in many cell systems has been shown to be associated with an increase in oxidative stress. Oxidative stress signalling is known to act as a signalling mediator of biological responses upon exposure to various physical and chemical agents. Recently, its role as a bystander signalling mediator has been proposed. DNA is vulnerable to reactive oxygen species (ROS)-induced damage [18]. We therefore hypothesize that TNF{alpha} secreted after radiation exposure can cause genetic damage by generating free radicals, and that this genetic damage may pass on to successive generations resulting in genomic instability. This study was carried out in primary vascular endothelial cells, a cell type that is exposed to radiation more frequently than other cell types and can communicate rapidly with other tissues [21].


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
Human primary aortic endothelial cells (HAECs; Clonetics, San Diego, CA) were cultured as described previously [19]. Briefly, cells were grown at 37°C in a 5% CO2/air incubator in MCDB-131 medium (Sigma, St Louis, MO) containing 10% fetal bovine serum (Hyclone, Kansas City, KS), 1 ng ml–1 hydrocortisone (Sigma, St Louis, MO) and 100 U ml–1 penicillin/streptomycin (Mediatech, Herndon, VA). The medium was enriched with 250 ng ml–1 fibroblast growth factor and 1 µg ml–1 epidermal growth factor (PeproTech, Rocky Hill, NJ). Fresh complete medium was replaced every 2–3 days. Culture passages from 4 to 7 were used in all experiments.

Exposure to ionizing radiation
For the selected doses of low-LET radiation exposures, the cells were removed from a 37°C incubator and exposed to 0.1 Gy, 1 Gy or 2 Gy of 137Cs {gamma}-rays at a dose rate of 1.29 Gy min–1 (Atomic Energy of Canada Ltd GammaCell-40 Irradiator) or the MRC 14 mAX–250 KeV X-ray source (Siemens) at Harwell, UK, at room temperature (~ 22°C). Immediately after exposures, the cultures were returned to the 37°C incubator and harvested at selected time points specified for each experiment. Mock-irradiated control cells (0 Gy) were treated identically.

Treatment
All antioxidants and the antioxidant enzymes were purchased from Sigma Chemical Co., St Louis, MO. Stock solutions (1000 U µl–1) of catalase, polyethylene glycol-tagged superoxide dismutase (PEG-SOD) and polyethylene glycol-tagged catalase (PEG-CAT) made in distilled water were flushed with argon gas and stored in amber-coloured bottles at –20°C. A final concentration of 1000 U ml–1 catalase, 600 U ml–1 PEG-SOD and 300 U ml–1 PEG-CAT was added directly into the culture medium. Stock solutions (0.5 M) of pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) were prepared in Hanks' balanced salt solution. Because NAC in solution is acidic and thus alters the pH when added to culture medium, the stock solution of NAC was adjusted to pH 7.4 by the addition of 3 N sodium hydroxide [3]. PDTC and NAC were used at final concentrations of 150 µM and 20 mM, respectively, in culture. Care was taken to avoid photosensitization of these antioxidant reagents by performing the experiment under yellow light. Catalase, PDTC and NAC were added to cultures 1 h prior to exposure to either X-rays or exogenous TNF{alpha}. PEG-SOD and PEG-CAT were added into the culture and incubated for at least 16 h before TNF{alpha} treatment.

Cell viability
Cell viability was determined by the trypan blue dye exclusion method. Cells at a density of 1 x 105 cells ml–1 were seeded on to 6-well plates (Corning, NY) and cultured in complete growth medium. When the cells were 70% confluent, they were treated with TNF{alpha} at final concentrations of 0.1 ng ml–1, 1 ng ml–1 and 10 ng ml–1 for 5 h, at which point the medium was removed and the cells were covered with fresh medium. After 24 h, 48 h, 72 h and 96 h of incubation, the cells were harvested by trypsinization with 0.25% trypsin–EDTA (Invitrogen, Carlsbad, CA). The cell suspension was added with 0.04% trypan blue (Sigma, St Louis, MO), and cell viability was determined using a haemocytometer. Four samples were included in each experimental group. The fold growth was determined by examining population doubling time and normalized against the mock-treated control cells. For the clonogenic assay, the cells were seeded as two sets at 5 and 10 cells per well in duplicate in 96-well culture plates either immediately after exposure to 0.1 Gy, 1 Gy or 2 Gy or after 5 h incubation with 0.1 ng ml–1, 1 ng ml–1 and 10 ng ml–1 TNF{alpha}. The surviving fraction was estimated on day 14 by normalizing against the mock-treated control cells.

Apoptotic assay
For morphological examination of apoptotic cells, a dual staining method was followed. Cells (1 x 105 cells) grown on coverslips in a 6-well plate were incubated for 24 h with 0.1 ng ml–1, 1 ng ml–1 and 10 ng ml–1 TNF{alpha}. The cells were then washed and stained with a combination of the fluorescent DNA dyes, acridine orange (100 mg ml–1) and ethidium bromide (100 mg ml–1), and immediately observed under a fluorescence microscope (Nikon-ES 600) equipped with a digital camera (CoolPix-4500; Nikon Inc., NY) using an excitation wavelength of 450/490 nm and an emission wavelength of 520 nm. Four morphological states were examined: (1) viable cells with normal nuclei (bright green chromatin with organized structure); (2) viable cells with apoptotic nuclei (green chromatin which is highly condensed and/or fragmented); (3) non-viable cells with normal nuclei (bright orange chromatin with organized structure); and (4) non-viable cells with apoptotic nuclei (bright orange chromatin which is highly condensed or fragmented).

Enzyme-linked immunosorbent assay (ELISA)
TNF{alpha} expression was determined by sandwich ELISA. For each experiment, the cells were grown to near confluence and incubated for at least 16 h in growth factor-free medium containing reduced serum (2%) to eliminate any growth factor- or serum-induced non-specific effects. Endothelial cells (5 x 106 cells) at a density of 1 x 106 cells ml–1 were exposed to a total dose of 1 Gy or 2 Gy and harvested at 2 h, 4 h, 8 h, 16 h and 24 h. The level of secreted TNF{alpha} in 0.1 ml of culture supernatant was estimated using a Quantikine Cytokine Detection System following the manufacturer's protocol (R&D Systems, Minneapolis, MN). The values were extrapolated from the standard curve using human recombinant TNF{alpha} (hrTNF{alpha}). The induced levels of TNF{alpha} after radiation exposure were calibrated from running a hrTNF{alpha} standard curve using reagents supplied by R&D Systems. Intra-assay variation was determined by including a known amount (500 pg ml–1) of TNF{alpha} (as recommended by the manufacturer's protocol: R&D Systems low range calibration) for each run.

Measurement of intracellular oxidative stress
To measure the induced intracellular oxidative free radicals, the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Portland, OR) was used. The esterified form of dichlorofluorescein (dichlorofluorescein diacetate) crosses the cell membrane where it undergoes deacetylation by intracellular esterase. The resulting compound, dichlorofluorescein, is trapped within the cell and is therefore susceptible to radical-mediated oxidation. Increased fluorescence emission reflects enhanced oxidative stress. Endothelial cells at 70% confluency were incubated with DCFH-DA (10 µM) in phenol red-free medium for 30 min. In order to remove excess dye in the medium, the cells were washed thoroughly but gently with PBS and then treated with 0.1 ng ml–1, 1 ng ml–1 or 10 ng ml–1 hrTNF{alpha} (Biosource Inc., Camarillo, CA). The cells were incubated in the dark for 10 min, 30 min, 60 min and 120 min. In experiments in which PEG-SOD (600 U; Sigma) and PEG-CAT (300 U; Sigma) were used, the cells were incubated with the antioxidants for 16 h prior to TNF{alpha} treatment. Fluorescence images were acquired and photographed at 100x under fluorescein isothiocyanate (FITC) filter (488 nm) using a Nikon TE-2000U inverted microscope equipped with a digital camera (CoolPix-4500; Nikon Inc., NY). For quantitative measurements, the cells were trypsinized, washed in 1x PBS twice and quantified using an EPICS ELITE flow cytometer (Coulter Cytometry, Miami, FL) with a 15 mW air-cooled argon ion laser operated at 7 A and 488 nm. Histograms were analysed using Multicycle-Plus, version 3.0 (Phoenix Flow Systems, San Diego, CA). An aliquot of medium was removed before trypsinization to determine the baseline fluorescence levels. Cells pretreated with DCFH-DA and then treated with 100 µM H2O2 were used as positive controls.

Assessment of chromosome damage
Genomic instability was characterized by delayed chromosome aberrations as described previously [4]. The group was classified as "unstable" if levels of aberrations were significantly elevated over control levels. After exposure to 0.1 ng ml–1, 1 ng ml–1 or 10 ng ml–1 TNF{alpha} for 5 h or 0.1 Gy or 2 Gy X-rays, cell cultures were established with 10 ml of growth medium in 75 cm2 tissue culture flasks. At days 3, 12 and 20 (approximately 1–12 population doublings), cells were harvested for cytogenetic analysis. Coded chromosome preparations of the various cell populations were made by accumulating metaphases in the presence of 0.05 mg ml–1 colcemid for 2 h, followed by treatment with 0.05% potassium chloride (w/v) and fixation in methanol and acetic acid (3:1 v/v). Fixed cells were spread on slides, air dried and processed for conventional chromosomal analysis by solid Giemsa staining. To determine the frequency of karyotypical abnormalities, 50–100 well-spread metaphases per treatment group were analysed with a light microscope.

Comet assay
The comet assay was performed as described previously [20]. Partly frosted microscope slides were coated with 1% normal melting point agarose and allowed to dry. The slides were then placed on a metal tray on ice. Treated cell suspensions at 3 x 104 cells per slide were mixed 1:10 with 1% low melting point agarose, pipetted onto the slides and overlaid with a coverslip briefly until set. The slides were then placed into coplin jars filled with cold lysis buffer (2.5 M NaCl, 100 mM EDTA, pH 8.0, 10 mM Tris-HCl, pH 8.0, 1% Triton X-100 and 1% dimethyl sulphoxide, pH > 10). The jars were kept protected from light and placed into a 4°C refrigerator for at least 1 h or overnight. A horizontal electrophoresis tank was placed in a cold room (4°C) and filled with alkali buffer (0.3 M NaOH and 1 mM EDTA, pH 12). The slides were placed into the alkali buffer for 1 h prior to electrophoresis. Electrophoresis was performed at a constant voltage of 22 V (approximately 300 mA) for 30 min. The slides were then removed and rinsed three times for 10 min each with cold neutralizing buffer (500 mM Tris-HCl, pH 7.5). To determine the average %DNA in the comet tails, the slides were then immediately stained with a 1x solution of SYBR Gold (Molecular Probes/Invitrogen, Carlsbad, CA) in TE buffer and analysed with a fluorescent microscope using Komet 5.5 image analysis software (Kinetic Imaging Technology, South Windsor, CT).

Statistical analysis
For genetic analysis, variation between experiments within a group was assessed using the Mann–Whitney U-test. Experiments were grouped, and comparisons between groups were made by the Fisher's exact test. For biochemical assays, the significance was determined with Student's unpaired t-test. For all tests, a value of p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
TNF{alpha} secretion upon radiation exposure
In order to study whether exposure of endothelial cells to radiation could induce TNF{alpha} expression, the secreted protein levels in the culture supernatant were measured. The levels of secreted TNF{alpha} in culture supernatants from cells (1 x 106 cells ml–1) exposed to a single dose of 1 Gy or 2 Gy and harvested at 2 h, 4 h, 8 h, 16 h and 24 h were estimated by sandwich ELISA. In this cell type, the constitutive TNF{alpha} level was below 25 pg per 106 cells ml–1. In the mock-irradiated control cells, these levels did not change significantly at any time points examined. In the positive control, where cells were incubated with 1 µg ml–1 lipopolysaccharide (LPS) for 16 h, there was a profound increase in TNF{alpha} secretion. This indicates that the cells are in a state to respond to the stimulation of TNF{alpha} expression. Next, when the cells were irradiated, there was no increase in TNF{alpha} levels at 2 h, 4 h or 8 h post exposure compared with sham-irradiated control cells. An increase in the levels of secreted TNF{alpha} in both 1 Gy and 2 Gy exposed cells was first observed at 16 h. The levels were 270 and 230 pg per 106 cells ml–1, after 1 Gy and 2 Gy exposure, respectively (p ≤ 0.01). The TNF{alpha} secretion was persistently high in the culture supernatant harvested even at 24 h post exposure (Figure 1Go). It is interesting to note that the induced levels of TNF{alpha} remained at a similar level in both 1 Gy and 2 Gy exposure at all time points examined, suggesting that the secreted TNF{alpha} in the primary endothelial cells is independent of the total dose at which the cells are exposed. The results, however, clearly indicated that primary endothelial cells secrete TNF{alpha} protein in response to low doses of radiation exposure.


Figure 1
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  		Figure 1. Enzyme-linked immunosorbent assay (ELISA) measurement showing the time-dependent secretion of tumour necrosis factor (TNF){alpha} 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{alpha} was carried out in 0.1 ml of culture supernatant. Cell counts were performed before harvesting, and TNF{alpha} levels in picograms were normalized to 1 x 106 cells ml–1.

 
Cell viability/survival of endothelial cells in response to TNF{alpha}
To examine whether hrTNF{alpha} modulates cell survival, aortic endothelial cells (40 000 cells/T-25 flask) at passage five were incubated in duplicate with 0 ng ml–1, 0.1 ng ml–1, 1 ng ml–1 and 10 ng ml–1 hrTNF{alpha} (Figure 2aGo). Cells exposed to 0.1 Gy, 1 Gy and 2 Gy were used as positive controls (Figure 2bGo). Survival (measured as fold growth) was estimated at 24 h, 48 h, 72 h and 96 h after exposure to either X-irradiation or TNF{alpha}. Clonogenic survival was carried out in 96-well plates and examined on day 14. TNF{alpha} at the concentrations used in these studies caused only minimal or no cytotoxicity. The cell survival measured by population doubling at 96 h showed a 1.42 ± 0.13-, 1.48 ± 0.15- and 1.18 ± 0.11-fold increase at 0.1 ng ml–1, 1 ng ml–1 and 10 ng ml–1 concentrations, respectively (Figure 2aGo). Cells exposed to the lowest dose of 0.1 Gy X-rays also showed an increase at 1.29 ± 0.15, which is not unusual and can be explained by a temporary response following a low dose of ionizing radiation. However, cells exposed to 1 Gy and 2 Gy showed an increased cell loss at 0.69 ± 0.22 and 0.51 ± 0.05, respectively. Similar results were observed with the clonogenic assay measured after 14 days. The surviving fractions at 0.1 ng ml–1, 1 ng ml–1 and 10 ng ml–1 TNF{alpha} showed a 1.26 ± 0.14-, 1.48 ± 0.27- and 1.29 ± 0.49-fold increase in cell densities, respectively. The surviving fractions at 0.1 Gy, 1 Gy and 2 Gy compared with sham-irradiated controls were 1.4 ± 0.16, 0.57 ± 0.01 and 0.48 ± 0.03, respectively (Figure 2bGo).


Figure 2
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  		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){alpha} 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{alpha} 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.

 
Apoptosis
Cells predisposed to apoptosis will shrink and exhibit chromatin condensation and, subsequently, internucleosomal cleavage of DNA. Dual staining and examination of the morphological characteristics of cells treated with TNF{alpha} at concentrations of 0.1 ng ml–1, 1 ng ml–1 or 10 ng ml–1 revealed bright green fluorescence with homogeneous chromatin staining pattern (Figure 2cGo). The pattern was similar to that of untreated control cells in which the cells were viable with normal nuclei. At a higher concentration of TNF{alpha} (10 ng ml–1), most of the cells showed a characteristic normal pattern with a very minimal orange fluorescence, indicating the occurrence of a few apoptotic cells. In contrast, cells treated with staurosporine (10 µM; Sigma) showed swelling of nuclei with complete chromatin condensation and bright orange fluorescence representing apoptotic or non-viable cells with apoptotic nuclei.

Intracellular oxidative stress in primary endothelial cells treated with TNF{alpha}
To assess the TNF{alpha}-induced intracellular oxidative burst, the oxidation-sensitive probe DCFH-DA was used. This is a reliable assay for the measurement of global intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH•) and hydroperoxides (ROOH). There was no detectable DCFH fluorescent signal even after 120 min in sham-treated cells. Endothelial cells treated with 100 µM H2O2 (positive control) revealed an increase in fluorescence within 10 min. TNF{alpha} significantly increased ROS production similar to that of the positive control. Cells treated with 10 ng ml–1 TNF{alpha} showed increased fluorescent signals as early as 10 min. This increase in the production of ROS was completely blocked when cells were preincubated with PEG-SOD and PEG-CAT, confirming that the fluorescent signal upon TNF{alpha} treatment was due to increased production of ROS (Figure 3aGo). Quantitative analysis, measured by flow cytometry of endothelial cells incubated with increasing concentrations of TNF{alpha}, showed a dose-dependent increase in the fluorescent intensities. The levels at 0.1 ng ml–1 and 1 ng ml–1 TNF{alpha} were 18.53 ± 0.91 and 79.14 ± 2.24. However, at 10 ng ml–1, the fluorescent intensity remained at the same or slightly reduced level (69.32 ± 3.61) compared with 1 ng ml–1 treated cells, indicating that the production of ROS reached a saturation level at 1 ng ml–1 TNF{alpha} (Figure 3bGo). These results strongly support our hypothesis that TNF{alpha} at concentrations at which no toxicity was observed could potently induce free radicals in vascular endothelium.


Figure 3
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  		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){alpha} 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{alpha}-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{alpha}, (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.

 
Chromosome damage induced by TNF{alpha}
Flasks counted at first passage to determine cell survival were subsequently harvested for chromosomal analysis at day 3 post treatment. In addition, cultures were maintained and harvested at day 12 and day 20 for chromosomal analysis. Prepared metaphases were solid stained with Giemsa, and a minimum of 50 were analysed for chromosome- and chromatid-type aberrations, indicative of damage that has occurred within the last cell cycle. These data were obtained from three to five independent experiments for each radiation dose or TNF{alpha} concentration at days 3, 12 and 20. In each experiment, pooled duplicate cultures were analysed for each sample. At the completion of scoring, treatments were assigned to the coded data, and differences between the fractions of aberrant cells from each harvesting point were ascertained by the Fisher's exact test. Significant differences between the various classes of aberration types were confirmed by a conditional binomial test. At day 3 (approximately first passage), all treated groups showed significant (p ≤ 0.05) chromosomal damage compared with the control. Interestingly, this damage was maintained in all treated groups at day 12 (approximately seven cell doublings) and at day 20 (approximately 12 cell doublings) (Figure 4Go). These data indicate that TNF{alpha} treatment alone is capable of inducing persistent genomic instability at levels comparable with radiation exposure, which has been observed here and in other studies [39, 40].


Figure 4
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  		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){alpha} 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.

 
TNF{alpha}-induced DNA damage is attenuated by antioxidants
The single-cell gel electrophoresis assay (comet) was used to examine the extent of DNA damage. The comet assay is preferred because this is a sensitive and reliable method of measuring both the initial levels of radiation-induced DNA damage and the repair kinetics at the level of single cells. A major advantage of using this method is that the assay requires only a limited number of cells to determine either single or double strand breaks. To determine the extent of genetic damage from a 5 h incubation of TNF{alpha} in human aortic endothelial cells, the comet assay was performed immediately following treatment, and genetic damage was estimated by measuring the percentage of DNA content in the comet tail. The negative control cells showed a basal level of 10.9 ± 0.56% (Figure 5aGo). The cells exposed to TNF{alpha} at the lowest concentration of 0.1 ng ml–1 showed a significant (p ≤ 0.001) increase in damage (14.2 ± 0.81%). Both the higher concentrations of TNF{alpha} (1 ng ml–1 or 10 ng ml–1) increased the damage twofold (22.8 ± 1.3% and 20 ± 0.87%, respectively) compared with controls. Similarly, 2 Gy exposed cells showed an increase of 16.3 ± 0.6%. The positive control H2O2 treatment showed the maximum damage of 48.1 ± 0.74%. These results indicate that TNF{alpha} was not only capable of causing genetic damage, but also at higher levels compared with 2 Gy (p ≤ 0.001).


Figure 5
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  		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){alpha}. Cells exposed to 2 Gy or treated with 100 µM H2O2 were used as positive controls. Cells were treated with TNF{alpha} 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{alpha}-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{alpha} 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{alpha} or exposure to X-rays or H2O2.

 
To confirm that the TNF{alpha}-induced genetic damage is due to an increase in intracellular free radicals, endothelial cells were enriched with antioxidant enzymes or free radical neutralizing compounds before TNF{alpha} treatment. The cells were then examined for immediate and persistent genetic damage by estimating the percentage of DNA content in the comet tail. Cells preincubated with 1000 units ml–1 antioxidant enzyme catalase significantly blocked the occurrence of DNA damage caused by TNF{alpha}, radiation or H2O2. With antioxidant pretreatment, the percentage of comet tail DNA in 10 ng ml–1 TNF{alpha}, 2 Gy X-ray or 100 µM H2O2 treated cells was attenuated to 7.3 ± 0.7, 6.2 ± 0.6 and 11.7 ± 0.9, respectively, in the presence of catalase (Figure 5bGo). Similarly, the metal chelator PDTC and glutathione precursor NAC markedly decreased the DNA damage caused by 10 ng ml–1 TNF{alpha}, 2 Gy X-rays or 100 µM H2O2. After pretreatment with PDTC and NAC, the percentage of comet tail DNA in TNF{alpha}, radiation and H2O2 treated cells was attenuated to 8.7 ± 1.3, 8.4 ± 1.1 and 14.9 ± 1.2, respectively (Figure 5cGo). These results revealed that the occurrence of genetic damage immediately after TNF{alpha} treatment is through an oxidation-dependent mechanism.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The contribution of TNF{alpha} to the initiation of the bystander effect and genomic instability has not been investigated directly. In our previous study, a significant decrease (about 60%) in genomic instability was observed in irradiated human lymphocytes when incubated with TNF{alpha} antibody prior to radiation exposure [9]. These results clearly indicated the possible role of TNF{alpha} in influencing genetic stability. In the present study, we have examined the mechanism involved in TNF{alpha}-induced genomic instability. We have demonstrated that, when added ectopically, TNF{alpha} induces free radicals, which may be responsible for the associated DNA damage. This TNF{alpha}-induced DNA damage was significantly blocked by the antioxidant enzyme catalase or the antioxidant compounds PDTC and NAC. Furthermore, this DNA damage is sustained up to several generations, indicating the occurrence of genomic instability in HAECs. This is in accordance with our observation that, following exposure to ionizing radiation, HAECs will secrete TNF{alpha} in a delayed manner with detection above endogenous levels starting at 8 h and continuing up to 24 h post irradiation.

Similarly, several studies have independently demonstrated the secretion of TNF{alpha} in endothelial cells in response to various stimuli [22–25]. Suschek et al [25] showed that vascular endothelial cells exposed to ultraviolet B (UVB; 10 mJ cm–2) radiation secreted maximum levels of TNF{alpha} in the culture supernatant at 18 h and 24 h. In addition, it has been shown previously that induced levels of TNF{alpha} in the culture medium are capable of inducing oxidative stress in target cells [26, 27]. In our study, cells treated with TNF{alpha} at concentrations ranging from 1 ng ml–1 to 10 ng ml–1 showed a maximum signal at 30 min. Suematsu et al [28] have shown, in cardiac myocytes, a similar rapid increase (as early as 1 h) in the production of ROS by TNF{alpha} at the same concentration used in our study (10 ng ml–1). This increase was found to persist for as long as 24 h. On the basis of these results, it could be speculated that, unlike H2O2, cells once triggered with TNF{alpha} stimulation initiate cyclic processes that persistently maintain the elevated levels of ROS. This prompts a further understanding of the mechanism of TNF{alpha}-induced ROS generation. In this context, several pathways have been suggested. In most cases, mitochondria have been identified as a major source of TNF{alpha}-induced ROS production, particularly in tumour cells, hepatocytes and vascular endothelial cells [27, 29, 31]. Evidence showing the inhibition of TNF{alpha} (10 ng ml–1)-induced DCFH oxidation by the mitochondrial respiratory chain inhibitor rotenone indicates that impaired mitochondrial electron transport activity may be the major source of TNF{alpha}-induced ROS production [28]. Alternatively, TNF{alpha} may exert a negative influence on the levels of intracellular glutathione [32] and an increase in lipid peroxidation and could, thereby, stimulate endothelial cell oxidative stress.

Oxidative stress has been suggested as being responsible for the increased presence of sister chromatid exchange in normal human cells [11]. TNF{alpha} initiates DNA damage as early as 3 h after its addition to cell culture [33]. Cells with DNA damage caused by TNF{alpha} are diverted from the pro-apoptotic pathway, thus allowing the cells with genetic damage to survive and pass on damage to successive generations, resulting in genomic instability. It is interesting to note that TNF{alpha} at the concentrations used in this study did not induce a pro-apoptotic pathway or affect the cell viability within 96 h or cell survival even after 14 days. Endothelial cells, other than in tumour angiogenesis, are normally resistant to TNF{alpha}-induced cell death [34, 35]. Other studies have shown that endothelial cells treated with TNF{alpha} at concentrations as high as 100 ng ml–1 [36] did not show toxicity. Wheelhouse et al [38] have reported an associated increase in oxidative stress that resulted in the formation of 8-oxo-deoxyguanosine (8-oxodG) upon stimulating primary hepatocytes with TNF{alpha} without any subsequent loss of cell viability. The unresponsiveness of vascular endothelial cells to TNF{alpha} treatment at concentrations at which other cell types undergo rapid apoptosis may be attributed to their intrinsic capacity to induce specific anti-apoptotic responses involving protein kinase C-, PI3 kinase- and p38 kinase-dependent mechanisms [34]. Alternatively, TNF{alpha} may induce the activation of nuclear factor (NF)-{kappa}B with subsequent transactivation of the cytoprotective gene A1 [37]. These results clearly indicate that TNF{alpha}, at concentrations at which no cytotoxicity is observed, could induce genetic damage through free radical generation.

It can be argued that ROS induced by a single acute chemical exposure may not be important in initiating instability. Morgan et al [41] reported an absence of chromosomal instability in more than 80 independently isolated clones of GM10115 cells that were treated with H2O2 at concentrations that cause up to three logs of cell killing [42] or xanthine/xanthine oxidase at concentrations that cause up to four logs of cell killing [43]. It is highly likely that repeated exposure to chemical agents such as TNF{alpha} might be necessary to result in such delayed responses. In support of this hypothesis, it has been shown in other model systems that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress could cause gene amplification and genomic instability [44]. In the present study, we have shown that human endothelial cells, when exposed to a single acute dose of TNF{alpha} at 10 ng ml–1 concentration where minimal cell death was observed, can cause genetic damage that can be carried over to several generations. This observation appeared to be contradictory to the theory of null response to a single acute dose of chemical stimulus and can be explained as follows. The response to TNF{alpha} in our study, although seemingly an acute chemical stimulus, could initiate a cyclic process through which it can subject the cells to continuous exposure to free radicals. TNF{alpha} stimulates a number of cellular responses through binding with its receptors [22, 23]. These receptors initiate a number of downstream events, including the activation of the transcription factor called NF-{kappa}B [45, 46]. As illustrated in the flow chart (Figure 6Go), TNF{alpha}, while generating free radicals and causing DNA damage, may simultaneously initiate the activation of NF-{kappa}B [30] by signalling through its receptors. NF-{kappa}B, on activation, can in turn transactivate TNF{alpha} gene expression. Thus, the secreted TNF{alpha} could sustain the production of free radicals. This recurrent positive feedback cycle may be responsible for maintaining elevated levels of endogenous free radicals that may subsequently lead to genomic instability. This phenomenon is currently being investigated in our laboratory. The findings from this study could provide insight into mechanisms that regulate the induction and/or perpetuation of non-targeted effects of radiation exposure.


Figure 6
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  		Figure 6. The possible mechanism of how radiation-induced tumour necrosis factor (TNF){alpha} could cause genomic instability in the non-targeted cells as a bystander response.

 
This project was supported by the Office of Sciences (BER), US Department of Energy, Grant (No. DE-FG03-02ER63449) funding to M Natarajan.

Received for publication May 12, 2006. Revision received February 21, 2007. Accepted for publication March 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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