Hypoxia/reoxygenation-induced mutations in mammalian cells detected by the flow cytometry mutation assay and characterized by mutant spectrum

Abstract

Under hypoxic conditions, cells are more resistant to cell killing by ionizing radiation by a factor of 2.5 to 3, potentially compromising the efficacy of radiotherapy. It has been shown recently that hypoxic conditions alone are sufficient to generate mutations in vitro and in vivo, likely due to the creation of reactive oxygen species (ROS) and a decrease in mismatch and homologous recombination DNA repair activity. These factors are known precursors to the onset of genetic instability and poor prognosis. We have previously characterized the flow cytometry mutation assay and its sensitivity to detect significant mutant fractions induced by genotoxic agents that are not detected by other mammalian assays. Here we measure the mutant fraction induced by hypoxia. CHO A(L) cells cultured at <0.1% O(2) for 24 h generated a significant mutant fraction of 120 x 10(-5) and had growth kinetics and survival characteristics similar to those obtained with other mutagens. We investigated the role of ROS by treating cells with the radical scavenger DMSO, which significantly reduced hypoxia toxicity and mutagenesis. Single cells were sorted from the mutant population, and the resulting clonal populations were stained for five antigens encoded by genes found along chromosome 11 to generate mutant spectra. The mutations were primarily large deletions, similar to those in background mutants, but the frequency was higher. We have demonstrated that hypoxic conditions alone are sufficient to generate mutations in mammalian cells in culture and that the spectrum of mutations is similar to background mutations.

FIG. 1

Mutant yield (mutants/1 × 10⁵ cells) using the standard CHO A_L mutation assay for cells cultured under hypoxic conditions (<0.1–2.5% oxygen) for 1 to 7 days (minimum of three experiments run in triplicate). Mutant yield is corrected for background, and the error bars represent the standard error of the mean.

FIG. 2

Top: Example histogram of CHO A_L cells stained with PE-conjugated monoclonal antibodies against CD59 12 days after a 24-h <0.1% oxygen hypoxia treatment. Gating is set as 1% of the mean of the positive peak for hypoxia-treated cells. Bottom: Mutant yield (mutants/1 × 10⁵ cells) for control cells and cells cultured in <0.1% oxygen for 24 h. Also shown are the effects of 0.5% DMSO in F12 medium. Results are from five experiments run in triplicate (**P < 0.01), and the error bars represent the standard error of the mean.

FIG. 3

Survival of control cells and cells cultured for 24 h at <0.1% oxygen, then incubated in normal oxygen for 12 days. Also shown are the effects of 5% DMSO in F12 medium added 2 h before hypoxia and continued for 12 h after reoxygenation (two experiments run in triplicate, *P < 0.05). Error bars represent the standard error of the mean.

FIG. 4

Mutation spectra of chromosome 11 generated by flow cytometry analysis of five surface antigens for clones isolated 12 days after treatment from the 1% CD59 mutant region of (top) control cells and (bottom) hypoxia-treated cells. Missing antigens are represented by minuses while the remaining antigens are marked by pluses/shaded regions and measurements are relative to the terminus of the p-arm of chromosome 11.