Induction of chromosomal instability by chronic oxidative stress

Abstract

Earlier studies using GM10115 cells analyzed the capability of different DNA-damaging agents to induce genomic instability and found that acute oxidative stress was relatively inefficient at eliciting a persistent destabilization of chromosomes. To determine whether this situation would change under chronic exposure conditions, the human-hamster hybrid line GM10115 was cultured under conditions of oxidative stress. Chronic treatments consisted of 1-hour incubations using a range of hydrogen peroxide (25-200 microM) or glucose oxidase (GO; 5-50 mU/ml) concentrations that were administered once daily over 10 to 30 consecutive days. The toxicity of chronic treatments was modest (- one log kill) and consistent with the low yield of first division aberrations (<5%). However, analysis of over 180 clones and 36,000 metaphases indicated that chronic oxidative stress led to a high incidence of chromosomal instability. Treatment of cells with 100 and 200 microM hydrogen peroxide or 50 mU/ml GO was found to elicit chromosomal instability in 11%, 22%, and 19% of the clones analyzed, respectively. In contrast, control clones isolated after mock treatment did not show signs of chromosomal destabilization. These data suggest that chronic oxidative stress constitutes a biochemical mechanism capable of disrupting the genomic integrity of cells.

Figure 1

Standard curve for the measurement of H₂O₂. The 1.0, 10, and 100 mM H₂O₂ stock solutions were prepared and the actual concentration of these solutions was calculated based on its absorbance at 230 nm (ɛ=81/cm/mol). Aliquots of H₂O₂ (200 µl) were added to 800 µl of PBS containing o-DD (80 µg/ml) and 5 U of HRP (squares) or no HRP (circles). Samples were incubated at 37°C for 1 hour before measuring absorbance at 470 nm. Plots shown are the linear regression fits through all data derived from three independent experiments (±SD).

Figure 2

Generation of H₂O₂ by GO. GO treatment for 1 hour was made in serum-free DMEM in the presence (∼ 1x 10⁶, squares) or absence (circles) of cells. The background generation of H₂O₂ in cells was also determined (triangles). Following a range of GO treatments, samples were removed and assayed forH₂O₂ using the o-DD/HRP assay. Data shown were averaged from duplicate samples taken from each of two independent experiments (±SD).

Figure 3

Kinetics of H₂O₂ production and metabolism. GO (25 mU/ml) was added to serum-free DMEM in the presence (∼1x10⁶, squares) or absence (circles) of cells. H₂O₂ (200 µM) was also added to cultures of 1x10⁶ cells in serum-free DMEM as a comparison (triangles). Following the addition of GO or H₂O₂, aliquots were removed over the course of an hour and assayed for H₂O₂ content using o-DD and HRP. Plots shown were averaged from duplicate samples taken from a typical experiment.

Figure 4

Chronic levels of H₂O₂ exposure. GM10115 cells subjected to chronic oxidative stress were assayed periodically for the determination of H₂O₂ levels in culture. Triplicate samples removed on days 5, 15, and 30 were analyzed for H₂O₂ content by the o-DD/HRP assay, and values were converted to H₂O₂ concentration using the standard curve shown in Figure 1. For each day, increased shading of individual bars (from left to right) corresponds to 5, 10, 25, and 50 mU/ml GO or 25, 50, 100, and 200 (day 5 only) µM H₂O₂. Bar charts indicate the average of triplicate measurements (±SD).

Figure 5

Cell survival after chronic oxidative stress. GM10115 cells subjected to 30 consecutive days of GO or H₂O₂ exposure were plated for the determination of clonogenic survival. Surviving fraction was normalized to sham-treated controls set to unity. Data represent the average of three independent measurements (±SD).